Method for rapid identification of microorganisms

ABSTRACT

The present invention relates, in general, to probes, methods, and kits used to determine the presence or absence of a microorganism in a sample. The probes, methods, and kits comprise at least one capture probe and/or at least one detector probe.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/197,594, filed Aug. 5, 2005, which claims priority to U.S.Provisional Application Ser. No. 60/599,858, filed Aug. 10, 2004, thedisclosures of which are hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates, in general, to probes, methods, and kitsused to determine the presence or absence of a microorganism in asample. The probes, methods, and kits comprise at least one captureprobe and/or at least one detector probe.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Bacteremia and fungemia are life-threatening infections that requiretimely administration of appropriate antimicrobial therapy to preventsignificant mortality. The term “septicemia” is used to describe thepresence of organisms within the blood in association with laboratoryand/or clinical findings that are indicative of infection such as fever,chills, malaise, tachycardia, hyperventilation, shock and leucocytosis.Weinstein et al. (Rev. Infect. Dis. 5: 54-70 (1983)) determined that theoverall rate of mortality was 42% among 500 episodes of bacteremia andfungemia, with approximately half of the deaths attributable directly tosepticemia. It has long been recognized, however, that the majority ofbacteremias and fungemias are associated with the recovery of very lownumbers of organisms from the blood. Indeed, it is not uncommon for lessthan 1 organism/mL of blood to be present, particularly after theinitiation of antimicrobial therapy. The severity of such infections andthe diverse spectrum of potential pathogens, therefore, necessitatehighly sensitive methods of diagnosis that are capable of identifying abroad spectrum of bacteria and fungi. Classically, diagnosis is achievedthrough the use of broad-based culture methods that are amenable to thegrowth of a wide variety of pathogens from low-level inocula. Followinggrowth and isolation in pure culture, the organisms are identifiedthrough the application of a battery of biochemical tests. Antimicrobialsusceptibility testing is then conducted to permit modification ofempirical therapy to an efficacious pathogen-specific regimen thatminimizes cost and toxicity. There remains, however, a need to reducethe time between collection of specimens from a patient andadministration of targeted antimicrobial therapy to provide anopportunity to reduce morbidity and mortality, defray the cost oftherapy and hospitalization, and minimize the spread of antimicrobialdrug resistance caused by ineffective or inappropriate therapy.

SUMMARY OF THE INVENTION

The present invention relates to a method for identifying the presenceof at least one microorganism in a sample, the method comprising: (a)releasing RNA or DNA from the at least one microorganism in the sample;(b) contacting the RNA or DNA with at least one capture probe capable ofhybridizing to a first target sequence of the RNA or DNA, wherein thecontacting is performed under conditions that permit hybridizationbetween the first target sequence and the at least one capture probe toform a microorganism-capture probe hybrid complex, and wherein the atleast one capture probe comprises at least one sequence selected fromthe group consisting of SEQ ID NOs:1-53, 55, 56, 61, 62, 67, 68, and72-78; and (c) detecting the presence of the microorganism-capture probehybrid complex by (i) contacting the RNA or DNA with at least onedetector probe capable of hybridizing to a second target sequence of theRNA or DNA, wherein the detector probe comprises at least one reportergroup and wherein the contacting is performed under conditions thatpermit hybridization between the second target sequence and the at leastone detector probe to form a microorganism-capture probe-detector probehybrid complex, and wherein the at least one detector probe alsocomprises at least one sequence selected from the group consisting ofSEQ ID NOs:1-53, 55, 56, 61, 62, 67, 68, and 72-78; (ii) detecting themicroorganism-capture probe-detector probe hybrid complex by detectingthe at least one reporter group, wherein the presence of themicroorganism-capture probe-detector probe hybrid complex indicates thepresence of the at least one microorganism. In another embodiment, thereporter group is selected from the group consisting of a radioactiveisotope, an enzyme, a fluorescent molecule and an amplificationsequence. In a further embodiment, the amplification sequence initiatesan amplification reaction selected from the group consisting of stranddisplacement amplification (SDA), polymerase chain reaction (PCR),reverse transcriptase-strand displacement amplification (RT-SDA),reverse transcriptase-polymerase chain reaction (RT-PCR), nucleic acidsequence based amplification (NASBA), transcription-mediatedamplification (TMA), rolling circle amplification and Qβ replicaseamplification. In an additional embodiment, detection of themicroorganism-capture probe-detector probe hybrid complex isaccomplished via non-specifically labeling the hybrid complex.

In an additional aspect, the first target sequence and the second targetsequence comprise the same sequence. In another aspect, the captureprobe is immobilized on a solid support before hybridizing to the firsttarget sequence. In yet another aspect, the microorganism-capture probehybrid complex is immobilized on a solid support. In a further aspect,the microorganism-capture probe-detector probe hybrid complex isimmobilized on a solid support. In another aspect, the solid support isselected from the group consisting of latex beads, agarose beads,paramagnetic beads, ferric oxide, microarray chips, filter paper,nitrocellulose filters, nylon membranes, glass slides and cellularmembranes. In a further aspect, the solid support is a microarray chip.In an additional aspect, two or more capture probes are immobilized on asingle spot of the solid support. In a further aspect, the methoddescribed above further comprises an immobilization probe that iscapable of hybridizing to the capture probe to be immobilized onto thesolid support.

The methods of the present invention additionally provide a method foridentifying the species of one or more microorganisms in a sample, themethod comprising: (a) releasing RNA or DNA from the at least onemicroorganism in the sample; (b) contacting the RNA or DNA with at leastone species-specific capture probe capable of hybridizing to a firsttarget sequence of the RNA or DNA, wherein the contacting is performedunder conditions that permit hybridization between the first targetsequence and the at least one species-specific capture probe to form aspecies-specific microorganism-capture probe hybrid complex, and whereinthe at least one species-specific capture probe comprises at least onesequence selected from the group consisting of SEQ ID NOs:1-53, 55, 56,61, 62, 67, 68, and 72-78; and (c) detecting the presence of thespecies-specific microorganism-capture probe hybrid complex by (i)contacting the RNA or DNA with at least one detector probe capable ofhybridizing to a second target sequence of the RNA or DNA, wherein thedetector probe comprises at least one reporter group and wherein thecontacting is performed under conditions that permit hybridizationbetween the second target sequence and the at least one detector probeto form a species-specific microorganism-capture probe-detector probehybrid complex, and wherein the at least one detector probe alsocomprises at least one sequence selected from the group consisting ofSEQ ID NOs:1-53, 55, 56, 61, 62, 67, 68, and 72-78; (ii) detecting thespecies-specific microorganism-capture probe-detector probe hybridcomplex by detecting the at least one reporter group, wherein thepresence of the species-specific microorganism-capture probe-detectorprobe hybrid complex indicates the presence of the at least onemicroorganism belonging to the species. In a further embodiment, theamplification sequence initiates an amplification reaction selected fromthe group consisting of strand displacement amplification (SDA),polymerase chain reaction (PCR), reverse transcriptase-stranddisplacement amplification (RT-SDA), reverse transcriptase-polymerasechain reaction (RT-PCR), nucleic acid sequence based amplification(NASBA), transcription-mediated amplification (TMA), rolling circleamplification and Qβ replicase amplification. In another embodiment, thereporter group is selected from the group consisting of a radioactiveisotope, an enzyme, a fluorescent molecule and an amplificationsequence. In an additional embodiment, detection of themicroorganism-capture probe-detector probe hybrid complex isaccomplished via non-specifically labeling the hybrid complex.

In an additional aspect, the first target sequence and the second targetsequence comprise the same sequence. In another aspect, thespecies-specific capture probe is immobilized on a solid support beforehybridizing to the first target sequence. In yet another aspect, thespecies-specific microorganism-capture probe hybrid complex isimmobilized on a solid support. In a further aspect, thespecies-specific microorganism-capture probe-detector probe hybridcomplex is immobilized on a solid support. In another aspect, the solidsupport is selected from the group consisting of latex beads, agarosebeads, paramagnetic beads, ferric oxide, microarray chips, filter paper,nitrocellulose filters, nylon membranes, glass slides and cellularmembranes. In a further aspect, the solid support is a microarray chip.In an additional aspect, two or more species-specific capture probes areimmobilized on a single spot of the solid support. In a further aspect,the method described above further comprises an immobilization probethat is capable of hybridizing to the capture probe to be immobilizedonto the solid support.

The present invention further provides a method of determining theefficacy of an antimicrobial patient therapy, comprising: (a)identifying the presence or absence of a microorganism in a firstpatient sample according to the method claim 1; (b) identifying thepresence or absence of the microorganism in a second patient sampleaccording to the method of claim 1; wherein the first patient sample andthe second patient sample are taken sequentially over time, and whereindetection of the microbial nucleic acid in the first sample andsubsequent failure to detect nucleic acid in the second sample indicatesa successful response to therapy; and detection of the microbial nucleicacid in the second sample indicates the continued presence of viableorganisms in the sample. In an additional embodiment, the reporter groupis selected from the group consisting of a radioactive isotope, anenzyme, a fluorescent molecule and an amplification sequence. In anotherembodiment, the amplification sequence initiates an amplificationreaction selected from the group consisting of strand displacementamplification (SDA), polymerase chain reaction (PCR), reversetranscriptase-strand displacement amplification (RT-SDA), reversetranscriptase-polymerase chain reaction (RT-PCR), nucleic acid sequencebased amplification (NASBA), transcription-mediated amplification (TMA),rolling circle amplification, and Qβ replicase amplification. In afurther embodiment, the solid support is selected from the groupconsisting of latex beads, agarose beads, paramagnetic beads, ferricoxide, microarray chips, filter paper, nitrocellulose filters, nylonmembranes, glass slides and cellular membranes. In an additionalembodiment, the solid support is a microarray chip. In anotherembodiment, two or more capture probes are immobilized on a single spotof the solid support. In an additional embodiment, the method furthercomprises an immobilization probe that is capable of hybridizing to thecapture probe to be immobilized onto the solid support. In still anotherembodiment, detection of the microorganism-capture probe-detector probehybrid complex is accomplished via non-specifically labeling the hybridcomplex.

The present invention provides a kit for detecting the presence orabsence of at least one microorganism in a sample, comprising: (a) asolid support; (b) at least one capture probe comprising at least onecapture sequence capable of hybridizing to at least one target sequenceof RNA and/or DNA from the microorganism to form a microorganism-captureprobe hybrid complex; wherein the at least the detector probe alsocomprises at least one sequence selected from the group consisting ofSEQ ID NOs:1-53, 55, 56, 61, 62, 67, 68, and 72-78; (c) at least onedetector probe capable of hybridizing to a second sequence of the RNA orDNA, wherein the detector probe comprises at least one reporter group,and wherein the detector probe comprises at least one sequence selectedfrom the group consisting of SEQ ID NOs:1-53, 55, 56, 61, 62, 67, 68,and 72-78; and (d) a vessel to collect, concentrate, amplify or isolatethe RNA or DNA. In one aspect, the vessel is selected from the groupconsisting of evacuated blood collection tubes, eppendorf tubes and testtubes. In another aspect, the solid support is selected from the groupconsisting of latex beads, agarose beads, paramagnetic beads, ferricoxide, microarray chips, filter paper, nitrocellulose filters, nylonmembranes, glass slides and cellular membranes.

The present invention further provides an oligonucleotide for use indetecting a microorganism selected from the group consisting ofStaphylococcus aureus, Escherichia coli, Staphylococcus epidermidis,Klebsiella pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa,Streptococcus pneumoniae, Streptococcus mutans, Streptococcus gordonii,Clostridium perfringens, Clostridium botulinum, Haemophilus influenzae,Enterococcus durans, Streptococcus pyogenes, Streptococcus agalacticae,Clostridium difficile and Enterococcus faecium. In one embodiment,Staphylococcus aureus is selected from the group consisting of SEQ IDNOs:1, 2, 44, 47, 50, 61, 62, 73 and 76. In another embodiment,Escherichia coli is selected from the group consisting of SEQ IDNOs:3-7, 43, 46, 49, 52, 53, 55, 56, 72, 75 and 78. In a furtherembodiment, Staphylococcus epidermidis is selected from the groupconsisting of SEQ ID NOs:8-10, 45, 48, 51, 67, 68, 74 and 77. In anadditional embodiment, Klebsiella pneumoniae is selected from the groupconsisting of SEQ ID NOs:11-13. In yet another embodiment, Enterococcusfaecalis is selected from the group consisting of SEQ ID NOs:14-16. Inone aspect, Pseudomonas aeruginosa is selected from the group consistingof SEQ ID NOs:17 and 18. In another aspect, Streptococcus pneumoniae isselected from the group consisting of SEQ ID NOs:19 and 20. In a furtheraspect, Streptococcus mutans is selected from the group consisting ofSEQ ID NOs:21 and 22. In an additional aspect, Streptococcus gordonii isselected from the group consisting of SEQ ID NOs:23 and 24. In yetanother aspect Clostridium perfringens is selected from the groupconsisting of SEQ ID NOs:27 and 28. In another embodiment, Clostridiumbotulinum is selected from the group consisting of SEQ ID NOs:29 and 30.In a further embodiment, Haemophilus influenzae is selected from thegroup consisting of SEQ ID NOs:31 and 32. In an additional embodiment,Enterococcus durans is selected from the group consisting of SEQ IDNOs:35-37. In yet another embodiment, Streptococcus pyogenes is selectedfrom the group consisting of SEQ ID NOs:38-40. In a further aspect,Streptococcus agalacticae is selected from the group consisting of SEQID NOs:41 and 42. In another aspect, Clostridium difficile is selectedfrom the group consisting of SEQ ID NOs:25 and 26. In an additionalaspect, Enterococcus faecium is selected from the group consisting ofSEQ ID NOs:33 and 34.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts one embodiment of the use of the probes and methods ofthe present invention.

FIG. 2A depicts detection of a target oligonucleotide usingspecies-specific capture probes directly immobilized to a solid support.

FIG. 2B depicts detection of a target oligonucleotide usingspecies-specific capture probes immobilized to a solid support viaimmobilization probes.

FIG. 3 depicts an example of a solid support that may be used toimmobilize probes of the present invention and may be used in themethods and kits of the present invention.

FIG. 4 depicts a vessel capable of concentrating the microorganisms in asample, such as blood, that can be used in the methods and kits of thepresent invention.

FIGS. 5A-C depict exemplary capture probes according to the presentinvention immobilized to a different spots of an array usingimmobilization probes. Target oligonucleotides are bound to the captureprobes, and detector probes are bound to the target oligonucleotides.

FIG. 6 depicts synthetic target sequences derived from discontiguousregions within the ssrA (small stable RNA A) genes of E. coli, S.aureus, and S. epidermidis. Capture probes and detector probes that maybe used to capture and detect these sequences are also shown.

FIGS. 7A-E depict results from exemplary assays using methods describedherein using probes according to the invention.

FIG. 7F depicts the arrangement of immobilization probes and controls onchips according to the invention.

FIG. 8A depicts the arrangement on chips of capture probes according tothe invention and controls used in exemplary assays using methodsdescribed.

FIGS. 8B-E depict results from exemplary assays using methods describedherein using probes according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to probes, methods, and kits foridentifying the presence or absence of at least one microorganism in asample. The probes of the present invention comprise single-strandednucleic acid or nucleic acid derivatives such as a peptide nucleic acid.Probes of the present invention comprise (a) nucleic acid sequencescapable of hybridizing to nucleic acid sequences specific tomicroorganisms and/or (b) nucleic acid sequences capable of hybridizingto another probe according to the present invention.

Probes capable of binding microorganism RNA and/or DNA are referred toherein as “capture probes” and/or “detector probes.” Capture probes areoften, but need not be, immobilized to a solid support. Detector probesoften, but need not, comprise a means for facilitating detection of themicroorganism RNA and/or DNA. For example, detector probes often, butneed not, comprise a reporter group. Methods of the present inventioncomprise releasing RNA and/or DNA from at least one microorganism in asample and contacting the RNA and/or DNA with at least one capture probeunder conditions that permit specific hybridization between themicroorganism RNA and/or DNA and at least a portion of the probe to forma hybrid complex. A hybrid complex between a capture probe andmicroorganism RNA and/or DNA may be referred to herein as a“microorganism-capture probe hybrid complex.” The microorganism-captureprobe hybrid complex may, but need not, be detected with a detectorprobe that likewise forms a specific hybrid with the microorganism RNAand/or DNA. A hybrid complex between a detector probe and microorganismRNA and/or DNA that is also hybridized to a capture probe may bereferred to herein as a “microorganism-capture probe-detector probehybrid complex.” The presence or absence of a specific hybrid complexcorrelates with the presence or absence of the microorganism.

The probes and/or identification methods of the present invention may beused to identify the genus and/or species of one or more microorganisms.The probes and/or identification methods of the present invention may beused to determine whether one or more microorganisms of a particulargenus and/or species is present in a sample. Alternatively, the probesand/or identification methods of the present invention may be used toidentify whether a sample contains one or more microorganisms belongingto a general classification category such as a taxonomic family, or toeven a broader category. As a non-limiting example, the probes and/ormethods of the present invention can be used to determine whether asample contains a fungus, bacterium, virus, or parasitic microorganism.The probes and/or methods of the present invention may also be used todetermine susceptibility to antimicrobial agents by determining thepresence, absence and/or expression of specific markers, such as theantimicrobial drug resistance genes mecA, vanA or vanB.

For the purposes of the present invention, the term “microorganism” isused to mean a prokaryotic organism, a bacterium, a fungus, a parasite,a protozoan, or a virus. These terms are not mutually exclusive; as twonon-limiting examples, many protozoa are parasites, and all bacteria areprokaryotic organisms. A “sample,” as the term is used herein, can bederived from an animal and can include, for example, blood, urine orother body fluids, organs, tissues and any portions thereof, or can beobtained from the environment, such as air, water or soil, or frommaterial intended for human or animal use or consumption, such as meat,fish or dairy produce, and even cosmetics. Furthermore, the methods ofthe present invention may be performed on the entire sample or only aportion or fraction thereof. As a non-limiting example, a sample may bewhole blood from a subject, or the sample may be a collection ofplatelets isolated or concentrated from a subject's blood. As usedherein, “subject” means an animal. The term “animal” includes, but isnot limited to, birds, fish, and mammals, such as but not limited to,human and non-human primates, farm animals, and companion animals. Asused herein, the terms “subject” and “patient” are used interchangeably.The sample can also be derived from in vitro cultures of cells. Thecells of the cell culture can be eukaryotic or prokaryotic including,but not limited to, animal cells, plant cells, and bacterial cells. Thecell cultures can be, for example, derived cells isolated from tissues,organs, or body fluid of an animal or plant. In some embodiments, thebiological sample comprises animal cells that are derived from asubject. The term “sample” also encompasses a culture medium that hasbeen inoculated with a sample taken from a mammal, food, theenvironment, cosmetics, or the like to permit any microorganisms presentin the sample to replicate to detectable levels.

In certain embodiments, a sample is treated to concentrate or isolatemicroorganisms before releasing nucleic acid from them. Microorganismsmay be concentrated in a sample prior to, or simultaneously with, therelease of the nucleic acids. Alternatively, the DNA and/or RNA may bereleased prior to the concentration process.

Many methods for concentrating and/or isolating microorganisms are knownin the art. Examples of ways to concentrate the microorganisms in thebiological sample include, but are not limited to, using a WampoleIsolator™ tube (Wampole Laboratories, New Jersey, USA), a BD CPT™ tube(Becton, Dickinson and Company, New Jersey, USA), di-electrophoresis,traveling wave field migration, and electrophoresis. For example, FIG. 1illustrates one embodiment of the methods of the present invention inwhich bacteria in blood are concentrated into a volume of 300 μL using aWampole isolator tube. As another example, FIG. 4 depicts a vesselcapable of concentrating the microorganisms in a sample, such as blood,that can be used in the methods and kits of the present invention. Insome embodiments, a sample is treated to differentially separatemicroorganisms. In such embodiments, separate samples containingdifferent microorganisms may be obtained. Examples 1-3 hereinbelowdemonstrate differential separation of microorganisms using densitygradients and matrices.

As the present invention contemplates, concentration of microorganismsand/or their nucleic acids can be accomplished in any number of stepsincluding, but not limited to, one, two or three steps, where, in eachstep, the sample is progressively more concentrated. As a non-limitingexample, microorganisms or their nucleic acids may be first concentratedusing, for instance, a Wampole Isolator™ tube. To continue this example,the concentrated sample then may be concentrated further, separatingintact microorganisms or their nucleic acids using, for instance,electrophoresis.

As used herein, the terms “nucleic acids” and “oligonucleotides” areused to mean DNA or RNA, as is recognized in the art. Nucleic acids maybe single-stranded or double-stranded. Nucleic acids may be “released”from a microorganism using any means that will allow a capture probeaccess to hybridize the DNA or RNA. Hence, a “released” nucleic acid isa nucleic acid that is in a physical and chemical environment thatallows nucleic acid probes to bind to it. Additionally, both DNA and RNAmay be released by the same processes. Examples of ways that DNA or RNAmay be released include, but are not limited to, lysing themicroorganisms using, for example, heat, enzymes, detergents, buffers,acids, bases, chaotropes, physical shearing in the presence of beads orparticles and the application of pressure. As is contemplated by thepresent invention, the act of collecting, isolating, or concentratingthe sample, or the portion thereof to be tested, may sufficientlyrelease the nucleic acids that are subject to capturing. For thepurposes of the present invention, the DNA or RNA to be used may be, butneed not be, purified, isolated or concentrated further after release.Methods for purification, isolation, and concentration of nucleic acidsare well-known in the art. It is preferable that nucleic acids releasedfrom microorganisms be in single-stranded form and lack internalsecondary structure before they are contacted with probe(s) according tothe present invention. Accordingly, the methods of the present inventionmay also include one or more denaturing step, to denature anydouble-stranded nucleic acids or nucleic acids that possess internalsecondary structure that are released from the microorganisms, prior tocontacting the DNA or RNA with the capture probe. However, such adenaturing step is not required, particularly where the acts ofreleasing, purifying, isolating, and/or concentrating the nucleic acidsalso result in their denaturation. Methods for denaturation of nucleicacids are well-known in the art.

Microorganisms that are to be detected may be referred to as “targetmicroorganisms.” Nucleic acids released by microorganisms that are to bedetected may be referred to as “target nucleic acids” or “targetoligonucleotides.” Target oligonucleotides will usually comprise atleast one sequence that is capable of binding to a capture probe and/ora detector probe being used to detect microorganisms in a sample. Such asequence may be referred to herein as a “target sequence.”

Once nucleic acids are released from the microorganisms, they may becontacted with one or more capture probes that are immobilized on asolid support. As an alternative, the released nucleic acids may becontacted with a free (non-immobilized) capture probe to form a hybridcomplex, which is then contacted with a solid support that immobilizesthe hybrid complex. As yet another alternative, capture probes mayremain free (i.e., not immobilized). In such cases, hybrid complexes maybe isolated by art-known means such as electrophoresis.

As used herein, a “capture probe” is a nucleic acid, or nucleic acidderivative such as a peptide nucleic acid, that is capable of binding toa released nucleic acid. A capture probe contains at least onesingle-stranded portion, or sequence, that is capable of contacting andhybridizing with released microorganism nucleic acids. A sequence thatis capable of contacting and hybridizing with released microorganismnucleic acids may be referred to herein as a “capture sequence.” As usedherein, capture probes may be classified by, for example, themicroorganism(s) to which their capture sequence(s) is capable ofbinding. Thus, capture probes of the same “type” comprise capturesequence(s) capable of binding to the same microorganism(s). A captureprobe comprises at least one capture sequence. A capture probe oftenalso comprises, but is not required to comprise, sequences in additionto at least one capture sequence. Such additional sequences may, forexample, facilitate immobilization of the capture probe.

Detector probes may be used to facilitate the detection of nucleic acidsthat have been released from microorganisms. Nucleic acids that havebeen released from microorganisms may be contacted with one or moredetector probes. As used herein, a “detector probe” is a nucleic acid,or nucleic acid derivative such as a peptide nucleic acid, that iscapable of binding to a released nucleic acid and that is capable ofbeing detected, thereby facilitating detection of the released nucleicacid. A detector probe contains at least one single-stranded portion, orsequence, that is capable of contacting and hybridizing with a releasedmicroorganism nucleic acid. As with capture probes, a sequence of adetector probe that is capable of contacting and hybridizing withreleased microorganism nucleic acids may be referred to herein as a“capture sequence.” A detector probe is preferably bound to a reportergroup to facilitate detection. A detector probe to can be bound to areporter group before or after the detector probe is hybridized to atarget oligonucleotide. Reporter groups are known in the art and arediscussed in more detail hereinbelow.

The capture sequence of a detector probe will usually hybridize to adifferent target sequence of a target oligonucleotide than the targetsequence to which the capture sequence of the capture probe hybridizes.Accordingly, a target oligonucleotide can be bound and detected by adetector probe while is bound to a capture probe. In such embodiments,either the capture probe or the detector probe may be hybridized to thetarget oligonucleotide first. In certain embodiments it may be desirableto utilize a capture probe and detector probe each having a capturesequence that binds to the same target sequence. In such embodiments, atarget oligonucleotide may be captured by a capture probe, the captureprobe-target complex may be isolated, the capture probe-target complexmay be denatured, and the detector probe may them be hybridized to thetarget oligonucleotide.

As with capture probes, detector probes may be classified by, forexample, the microorganism(s) to which their capture sequence(s) iscapable of binding. Thus, detector probes of the same “type” comprisecapture sequence(s) capable of binding to the same microorganism(s). Adetector probe comprises at least one capture sequence. A detector probeoften also comprises, but is not required to comprise, sequences inaddition to at least one capture sequence. Such additional sequencesmay, for example, facilitate the binding of the detector probe to areporter group.

In some embodiments, capture probes and/or detector probes compriselinker molecules such as, but not limited to, carbon chains or nucleicacid sequences that are not complementary to the target oligonucleotide.A linker molecule may serve, for example, to attach a probe to a solidsurface, to bind a probe to another type of molecule (such as, forexample, a protein), to attach a probe to a reporter group, or to bind aprobe to another probe. A sequence that serves to immobilize a captureprobe to a solid surface may be referred to herein as an “immobilizationsequence.” A probe comprising an immobilization sequence may be referredto herein as an “immobilization probe.” Such non-complementary linkagesmay reduce steric hindrance and may also improve the kinetics ofhybridization by increasing the accessibility of the probes,particularly the capture sequence(s), to the bulk solution. Examples ofnucleic acid sequences that may be used as linker molecules include thehuman genes K-alpha (tubulin alpha-1), PPIA (peptidylprolyl isomeraseA), and UBC (ubiquitin-conjugating enzyme E2A), and portions thereof.FIGS. 5A-C provide non-limiting illustrations of capture probesaccording to the present invention immobilized to an array using linkerscomprising portions of K-alpha, PPIA, and UBC.

Capture probes of the current invention may be “immobilized” onto asolid support. As used herein, “immobilized” means affixed to a solidsupport such that movement of the capture probe in a solution islimited, i.e., a capture probe that is immobilized on a solid supportwill not dissociate from the solid support unless it is subjected to acondition or procedure that would cause it to dissociate.

As used herein, a “solid support” is a structure or a scaffold that willnot dissolve in a liquid or gas solution. Examples of solid supportsinclude, but are not limited to, latex beads, agarose beads, sepharosebeads, paramagnetic beads, ferric oxide, microarray chips, filter paper,nitrocellulose filters, nylon membranes, vessels, glass slides, and evencellular membranes. In some embodiments, the method of the presentinvention utilizes a three-dimensional microarray, such as, for example,the MetriGenix® Flow-Thru Chip® (MetriGenix, Inc., Maryland, USA), whichfacilitates increased hybridization kinetics. An example of aMetriGenix® Flow-Thru Chip® is illustrated in FIG. 3. Such porous arraysoffer increased surface area for attachment of probes over conventionaltwo-dimensional chips and permit the flow of liquid back and forth overthe chip surface of the array, thereby increasing the opportunity forcontact between the capture probe and target sequence. In someembodiments, each spot on an array may correspond to, as non-limitingexamples, a different species of microorganisms, group of microorganismsor epidemiological marker. In other embodiments, different types ofcapture probes may be immobilized on the same spot of an array. Multiplespots for each analyte or group of analytes may also be present.

Capture probes may be immobilized onto solid supports using any of themany art-known methods. Preferably, the immobilization does notadversely affect the capture probe's ability to bind to microorganismDNA and/or RNA or to other probes. A capture probe may be immobilizeddirectly to the solid support, or it may be immobilized indirectly viaattachment to another molecule that is immobilized on the solid support.For example, a capture probe may be immobilized using chemical or linkermoieties such as carbon chains or polyethylene glycol (PEG). In suchcases, the binding of the capture probe may be non-specific.Alternatively, methods of using chemical moieties to bind specificnucleic acid sequences are known and may be used with the presentinvention. As a non-limiting example, capture probes may bebiotinylated, with biotin possessing the ability to bind to avidin orstreptavidin. Continuing the example, the solid support may have avidinor streptavidin bound to it. Such a scheme is a non-limiting example ofa method for immobilizing capture probes without adversely affectingtheir ability to bind microorganism DNA and/or RNA because the biotincan be located at the opposite end of the molecule from the sequencecapable of binding microorganism DNA and/or RNA (which may be called a“capture sequence”), or the biotin may be located on an internal branchof the capture probe that will result in its being located at asufficient distance from the capture sequence that the binding of thecapture sequence to microorganism DNA and/or RNA is not hindered.

As another example, capture probes may also be immobilized using anothersingle-stranded oligonucleotide probe that is itself immobilized andthat is capable of hybridizing with the capture probe to be immobilized.Such oligonucleotide probes may be called “immobilization probes.” Theuse of immobilization probes is another non-limiting example of a methodfor immobilizing capture probes without adversely affecting theirability to bind microorganism DNA and/or RNA because the sequence on thecapture probe that is capable of binding to the immobilization probe(which may be called an “immobilization sequence”) can be located at theopposite end of the molecule from the sequence capable of bindingmicroorganism DNA and/or RNA (which may be called a “capture sequence”),or the immobilization sequence may be located on an internal branch ofthe capture probe that will result in its being located at a sufficientdistance from the capture sequence that the binding of the capturesequence to microorganism DNA and/or RNA is not hindered. An example ofimmobilization of a capture probe via an immobilization probe isillustrated in FIG. 2B. Examples of nucleic acid sequences that may beused as immobilization probes include the human genes K-alpha (tubulinalpha-1), PPIA (peptidylprolyl isomerase A), and UBC(ubiquitin-conjugating enzyme E2A). (FIGS. 5A-C). In an alternativeembodiment, immobilization probes may comprise non-specific sequencessuch as poly-A or poly-T oligomers. In a further embodiment they mayalso comprise random sequences of nucleotides or nucleotide homologueswith no homology or complementarity to naturally occurring nucleic acidsequences.

In some embodiments, capture probes may be immobilized directly orindirectly on a solid support in a pattern of discrete areas, or“spots.” Such a pattern, or a solid support capable of supporting such apattern, may be referred to herein as an “array,” a “microarray,” or a“chip.” The immobilization of probes of different types to a singlemicroarray or chip facilitates the simultaneous determination of whetherdifferent microorganisms are present in a single sample.

In certain embodiments, only a single type of capture probe isimmobilized to any one spot, and different types of capture probes maybe immobilized to different spots. In such embodiments, the identity ofthe capture probe immobilized in any given spot is known, somicroorganisms hybridized to capture probes in different spots can beidentified and differentiated from one another by means of theirlocations. Examples of immobilization of different types of captureprobes in different spots of arrays are illustrated in FIGS. 2A and 2Band in FIGS. 5A-C.

FIGS. 5A-C provide non-limiting exemplary illustrations of differenttypes of capture probes according to the present invention immobilizedto different spots of an array using immobilization probes andimmobilization sequences. Immobilization probes comprising approximately60 nucleotides in length to the human genes K-alpha (tubulin alpha-1)(FIG. 5A), PPIA (peptidylprolyl isomerase A) (FIG. 5B), or UBC(ubiquitin-conjugating enzyme E2A) (FIG. 5C) are immobilized to anarray. Each of the immobilization probes is hybridized to a captureprobe comprising approximately 30 bases of sequence complementary toK-alpha (FIG. 5A), PPIA (FIG. 5B), or UBC (ubiquitin-conjugating enzymeE2A) (FIG. 5C) 3′ to a capture sequence specific for E. coli (FIG. 5A),S. aureus (FIG. 5B), or S epidermis (FIG. 5C).

In other embodiments, more than one type of capture probe is immobilizedin a single spot. In such embodiments, it will often be useful to useemploy detector probes such that each detector probe of the same type isbound to a reporter group that is differentiable from reporter groupsbound to any other type of detector probe. As a non-limiting example,one could perform an assay in which detector probes that bind to atarget sequence from E. coli are labeled with fluorescein, and detectorprobes that bind to a target sequence from S. aureus are labeled withrhodamine. In such an assay, the presence of E. coli could bedifferentiated from the presence of S. aureus by the difference in thecolors of the fluorescent labels. Of course, detector probes withdifferentiable labels may also be used in conjunction with theimmobilization of different types of capture probes in different spots,thereby facilitating the performance of complex assays.

Detector probes may be attached directly or indirectly to a reportergroup. As an example of an indirect attachment using a linker molecule,a detector probe may comprise a “reporter adapter sequence” linker. Areporter adapter sequence is a portion of a detector probe that iscapable of binding via hybridization to a single-strandedoligonucleotide that bears a reporter group, which may be referred toherein as a “reporter probe.” FIGS. 2A-B provide exemplary illustrationsof a capture probe hybridized to a target oligonucleotide, which is inturn hybridized to a detector probe. The detector probe is hybridized toa reporter probe. In certain embodiments, detector probes of differenttypes may comprise the same reporter adapter sequence, therebyfacilitating the detection of different microorganisms using a singlereporter probe, which may be referred to herein as a “universal reporterprobe.” Such an embodiment is illustrated in FIGS. 2A and B. In otherembodiments, detector probes of different types may comprise differentreporter adapter sequences, thereby facilitating the use ofdifferentiable reporter probes to detect different microorganisms.

Capture probes and detector probes may be “protected” from prematurelyhybridizing to random nucleic acids by having a protecting groupsituated on or near the capture or detector probe. As non-limitingexamples, protecting groups include single-stranded nucleic acid that ispartially complementary to the capture probe to be protected, or anantibody or a binding fragment thereof that binds to the single-strandedportion of the capture or detector probe to be protected.

Hybridization between a microorganism nucleic acid and a capture probeor detector probe may be referred to herein as a “hybridization event.”A hybridization event will form a “hybrid complex.” As used herein, a“hybrid complex” is a double-stranded nucleic acid comprising at least aportion of a capture probe or detector probe (usually a capturesequence) and at least a portion of a target oligonucleotide (usually atarget sequence). A hybrid complex need not be double-stranded along itsentire length. Furthermore, for the purposes of the present invention, acapture probe or detector probe and a target oligonucleotide need nothave a complementary base pairing at every base for a hybridizationevent to occur. Further still, for the purposes of the presentinvention, a capture sequence and a target sequence need not have acomplementary base pairing at every base for a hybridization event tooccur. In other words, the present invention contemplates that a hybridcomplex will be formed even if a target oligonucleotide hybridizes to acapture or detector probe such that a portion of the capture or detectorprobe or target nucleic acid is single-stranded after hybridizationbecause the target oligonucleotide did not hybridize to the entirelength of the capture or detector probe. In some embodiments of thepresent invention, a portion of a capture or detector probe remainssingle-stranded after hybridization to a target oligonucleotide. In someother embodiments, a portion of a target oligonucleotide remainssingle-stranded after hybridization to a capture or detector probe. Instill other embodiments, portion(s) of each of a target oligonucleotideand a capture or detector probe remain(s) single-stranded afterhybridization to one another. A “portion” can be one or more nucleicacids in length. Such single-stranded portions may occur within and/oroutside of a capture sequence and/or a target sequence. Single-strandedportions within a capture sequence and/or target sequence may occur, asa non-limiting example, because the capture sequence and the targetsequence are not 100% complementary. Single-stranded portions outside ofa capture sequence and/or target sequence may occur, as a non-limitingexample, because the capture and/or detector probe contains portionsthat are not intended to bind to the target oligonucleotide.Single-stranded portions outside of a capture sequence and/or targetsequence may occur, as another non-limiting example, because the targetoligonucleotide comprises sequences in addition to the target sequence.For example, a capture probe will often (but need not) comprise asequence used to immobilize it to a solid support. As another example, adetector probe will often (but need not) comprise a sequence used tobind it to a reporter group. As yet another example, a targetoligonucleotide will often (but need not) comprise sequences 3′ and/or5′ to the target sequence(s).

A solid support may have immobilized to or on it one or variouscombinations of probes that are microorganism-specific, probes that arefor epidemiological markers (e.g., IS6110-based probes used forMycobacterium tuberculosis), and/or probes that are for drug resistancemarkers (e.g., mecA-based probes for methicillin resistance in S. aureusor rpoB-based probes for detection of rifampin resistance in M.tuberculosis). As used herein, the term “microorganism-specific probe”includes probes that are capable of hybridizing with a target sequencederived or released from a single microorganism species. Such probes mayalso be referred to herein as “species-specific probes.” The term“microorganism-specific probe” also includes probes that are capable ofhybridizing with target sequences from more than one species ofmicroorganism from a single genus of microorganism (e.g., IS6110 for thedetection of the M. tuberculosis complex (M. tuberculosis, M. bovis, M.microti, and M. africanum); probes based on conserved regions of the 16SrRNA, 18S rRNA, RNase P or ssrA gene sequences). For example, amicroorganism-specific probe may be designed to form a hybrid withnucleic acid sequences from both S. aureus and S. epidermidis, but notwith E. coli. Such probes may also be referred to herein as“genus-specific probes.” In some embodiments, a genus-specific probewill hybridize to sequences derived from all or many of themicroorganisms belonging to the same genus of classification. As usedherein, a “multi-genus probe” will hybridize to nucleic acid frommicroorganisms belonging to two or more different genera. A probe mayhybridize to an antimicrobial resistance marker that may be present inone or more species, for example. Such a probe may be species-specific,genus-specific, or multi-genus, depending on how widely theantimicrobial resistance marker is distributed through phylogeny.Whether a microorganism-specific probe, as contemplated by the presentinvention, hybridizes to a target sequence derived or released from asingle microorganism species, to target sequences derived or releasedfrom more than one microorganism species within the same genus ofmicroorganisms, or to target sequences derived or released frommicroorganisms from different genuses may also depend on thehybridization and wash conditions used in the assay.

As described above for capture probes, detector probes may bemicroorganism-specific probes, probes that are for epidemiologicalmarkers, and/or probes that are for drug resistance markers. Variouscombinations of capture probes and detector probes may be used todiscriminate between organisms present in a sample. For instance, agenus-specific capture probe may be used to immobilize microorganisms ofa selected genus, which then may be detected as a genus with one or moregenus-specific detector probes, or which may be discriminated by specieswith one or more species-specific detector probes. More than one type ofcapture probe may be used concurrently in the methods of the presentinvention Likewise, more than one type of detector probe may be usedconcurrently in the methods of the present invention.

As non-limiting examples, oligonucleotide probes comprising one or moreof the sequences set forth in the following table (Table 1) areparticularly useful for detecting and identifying bacteria of theindicated species. Oligonucleotide probes comprising one or more of thesequences set forth in Table 1 can be used as capture and/or detectorprobes to detect nucleic acids from the indicated bacterial species.Oligonucleotide probes comprising regions that are homologous to theoligonucleotide probes set forth in Table 1 are also useful forcapturing and/or detecting the indicated species. In general,oligonucleotides containing sequences that are at least about 85%, atleast about 90%, at least about 95%, or about 100% homologous to theoligonucleotides of Table 1 are useful.

The sequences in Table 1 can be used as species-specific capture and/ordetector sequences to detect and/or differentiate between particularspecies of microorganisms. Sequences from Table 1 may, but need not,comprise portion(s) of longer oligonucleotides. For example, probesaccording to the invention may comprise one or more sequences from Table1 and/or additional sequences.

TABLE 1 SEQ ID Reference Target Oligo 5′-3′ Tm NO: Species Strain GeneName Sequence (° C.)* Rank** 1 Staphylococcus NCTC ssrA S_aur-1 TTG ATT58.9 1 aureus 8325 AAG TTT CTT CTA AAC AGA 2 Staphylococcus NCTC ssrAS_aur-2 TCA TGA 59.7 1 aureus 8325 AAA GTG ATA AAC AAC C 3Escherichia coli O157:H7 ssrA E_coli-1 AAT TCC 59.2 2 EDL933 TAC GTCCTC GGT A 4 Escherichia coli O157:H7 ssrA E_coli-2 TAC ATT 60.0 2 EDL933CGC TTG CCA GC 5 Escherichia coli O157:H7 ssrA E_coli-3 CTA GCC 59.6 2EDL933 TGA TTA AGT TTT AAC G 6 Escherichia coli ATCC ssrA E_coli-4TCC TCG 59.8 2 133 GTA CTA CAT GCT TAG 7 Escherichia coli ATCC ssrAE_coli-5 TCC TAA 60.3 2 133 GAG CGG AGG CTA 8 Staphylococcus SR1 ssrAS_epi-1 CAT CAT 61.4 3 epidermidis GCT AAG CAA TAA ACA A 9Staphylococcus SR1 ssrA S_epi-2 TTG ATT 60.0 3 epidermidis ATA TTTCAT CTA AAC AGA CT 10 Staphylococcus SR1 ssrA S_epi-3 CAG TTA 61.2 3epidermidis TAT TTA ACC GAA ATG TGT 11 Klebsiella MGH ssrA K_pneu-1ATT CCT 60.1 4 pneumoniae ACA TCC TCG GCA 12 Klebsiella MGH ssrAK_pneu-2 GTC TTA 59.0 4 pneumoniae AGA GCG GAA GCT AG 13 Klebsiella MGHssrA K_pneu-3 AGC CTG 59.3 4 pneumoniae ATT AGA TTT AAC GC 14Enterococcus 775 ssrA E_faeca-1 CAT ATT 59.0 5 faecalis GCC ACT TAA ATCTCT AC 15 Enterococcus 775 ssrA E_faeca-2 CTG TAT 60.9 5 faecalisTGC TAG TCT GGT AAG CT 16 Enterococcus 775 ssrA E_faeca-3 ACA CTC 59.5 5faecalis ATT TAA AGG TTC GC 17 Pseudomonas ATCC ssrA P_aeru-1 GCT TAG59.4 6 aeruginosa 25330 CCA GCT CTA CTG AG 18 Pseudomonas ATCC ssrAP_aeru-2 TTA AGC 59.9 6 aeruginosa 25330 AGC TAG AGC GTA GTT 19Streptococcus Type 4 ssrA S_pneu-2 CTC AAG 59.4 8 pneumoniae TCT AGAAAC TGC GAG 20 Streptococcus Type 4 ssrA S_pneu-1 TTA TTT 60.6 8pneumoniae TAA CAG CCC CTC G 21 Streptococcus UA159 ssrA S_mut-1 TGT TTA59.9 10 mutans TTT AAC ACC GTT ACA AT 22 Streptococcus UA159 ssrAS_mut-2 TCA AAC 61.0 10 mutans TCT AAC GAT GCG AG 23 Streptococcus NotssrA S_gord-1 TGT TTT 60.7 10 gordonii Known AAC TTG ATT TTG ACA CA 24Streptococcus Not ssrA S_gord-2 CAA ATC 60.6 10 gordonii Known AAG CGAGTC TAT CAA 25 Clostridium 630 ssrA C_diff-1 CCA ACT 60.1 19 difficileTCA CTA ATA TCT CAC CT 26 Clostridium 630 ssrA C_diff-2 GTC CAG 59.6 19difficile TCT TAG TCG GCA G 27 Clostridium (Shimizu) ssrA C_perf-1AGC AGA 59.7 19 perfringens CCA GTA AGA CTT TCT AC 28 Clostridium(Shimizu) ssrA C_perf-2 AGA ACG 61.0 19 perfringens TCC ACA GAC AAA CTT29 Clostridium Hall A ssrA C_bot-1 AAC AGG 60.1 19 botulinum CTC CTAGAT TCA GTA G 30 Clostridium Hall A ssrA C_bot-2 CCG AGT 59.7 19botulinum GCA GTT TAT CCT T 31 Haemophilus Not ssrA H_infl-1 GAC ACG60.8 23 influenzae Known CTA AAC TTA AGC TAG TT 32 Haemophilus Not ssrAH_infl-2 CCT CAA 60.7 23 influenzae Known ACG GTG GCT TC 33 EnterococcusATCC ssrA E_faeci-1 GTC AAC 60.0 25 faecium 35667 TCA TTT AAG GAT TCA CT34 Enterococcus ATCC ssrA E_faeci-2 GAT GTT 60.5 25 faecium 35667CTC TTT TTC AAC TTA CAG 35 Enterococcus CNRZ129 ssrA E_dur-1 TCA ACT60.5 NR durans CAT TTG AGG TTT CG 36 Enterococcus CNRZ129 ssrA E_dur-2TGA TGA 60.8 NR durans TCT CTT TTA AAC TTT ACA G 37 Enterococcus CNRZ129ssrA E_dur-3 AGG CAT 60.6 NR durans TCT GTA TTG CTA GTC T 38Streptococcus M1 GAS ssrA S_pyo-1 TTA TGT 61.0 NR pyogenes SF370 CTT CATTTA ACA AAC TAA AG 39 Streptococcus M1 GAS ssrA S_pyo-2 TCA AGC 59.8 NRpyogenes SF370 CAT TAG TTT GCG 40 Streptococcus M1 GAS ssrA S_pyo-3GAC AAT 60.0 NR pyogenes SF370 TTC GTA ACC GTA GC 41 Streptococcus NCTCssrA S_agal-1 GTA TTG 60.8 NR agalacticae 8181 ATT TAA CTA GGT GAT GAC A42 Streptococcus NCTC ssrA S_agal-2 TTA ACT 60.5 NR agalacticae 8181AAC TAG ACA GTA GCC AAA C 43 Escherichia coli O157:H7 ssrA Eco_ssrA_DP50TCA GTC 75.0 2 EDL933 TTT ACA TTC GCT TGC CAG CTG CGG ACG GAC ACG CCACTA ACA AA 44 Staphylococcus NCTC ssrA Sau_ssrA_DP50 CTT CAA 70.0 1aureus 8325 ACG GCA GTG TTT AGC ATA TCC TAT TAA GGT TGA ATC GCG TTA AC45 Staphylococcus SR1 ssrA Sep_ssrA_DP50 CCA ACA 69.0 3 epidermidisTGA TAC TAG CTT GAT TAT ATT TCA TCT AAA CAG ACT TCA AGC GG 46Escherichia coli 0157:H7 rnp EcoCP4 GCA CTG 63.2 2 GTC GTG GGT TTC 47Staphylococcus WCUH29 rnp SauCP5 TTA CTC 59.9 1 aureus TAT CCA TAT CGAAAG ACT 48 Staphylococcus SR1 rnp SepCP6 CTA TTC 60.0 3 epidermidisTAA CCA TAT CCA ATG ACT 49 Escherichia coli 0157:H7 rnp Eco_rnp_DP50CCC CCC 77.0 2 AGG CGT TAC CTG GCA CCC TGC CCT ATG GAG CCC GGA CTT TCCTC 50 Staphylococcus WCUH29 rnp Sau_rnp_DP50 TAG GAT 67.0 1 aureusATT TCA TTG CCG TCA AAT TAA TGC CTT GAT TTA TTG TTT CAT CA 51Staphylococcus SR1 rnp Sep_rnp_DP50 TAG GTT 67.0 3 epidermidis ATT TCATTG CCG TCA AAT TAA TGC CTT GAT TTA TTG TTT CAT CA 52 Escherichia coliK12 16S EcoCP7 AGT GTG 59.0 2 rRNA GCT GGT CAT CCT 53 Escherichia coliRREC I 16S Eco_16_DP50 CTC AGA 75.0 2 rRNA CCA GCT AGG GAT CGT CGCCTT GGT GAG CCG TTA CCC CAC CAA CA *Nearest neighbor analysis **Rankingof species in top 25 (US) blood pathogens; NR = not ranked within top 25US blood pathogens

For the purposes of present invention, a capture probe captures (bybinding to) an oligonucleotide from a sample by hybridizing with it at asequence (e.g., a capture sequence) that is at least partiallycomplementary to a sequence (e.g., a target sequence) of theoligonucleotide being captured. Likewise, a detector probe detects (bybinding to) an oligonucleotide from a sample by hybridizing with it at asequence (e.g., a capture sequence) that is at least partiallycomplementary to a sequence of the oligonucleotide being captured (e.g.,a target sequence). As used herein, the phrase “partially complementary”means less than 100% complementary, but at least about 85%complementary. Accordingly, the phrase “at least partiallycomplementary” indicates that the capture sequence of a capture and/ordetector probe may between about 85% complementary to about 100%complementary to a target sequence to be useful according to the presentinvention. A capture sequence and a target sequence of anoligonucleotide to be captured may be, as non-limiting examples, atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, orabout 100% complementary to one another. For example, if a capturesequence is 100 bases long, and the target sequence is 95% complementaryto the capture sequence, the base pairs of the capture sequence and thetarget sequence will match in 95 of 100 bases of the capture sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% complementary to a target nucleic acidcan be determined conventionally using known computer programs such as,for example, the Bestfit program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, Wisconsin, USA). Bestfituses the local homology algorithm of Smith and Waterman, Advances inApplied Mathematics 2:482-489 (1981), to find the best segment ofhomology between two sequences. When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forexample, 95% complementary to a reference sequence according to thepresent invention, the parameters are set such that the percentage ofidentity is calculated over the full length of the reference nucleotidesequence, whether that be the capture probe or the target nucleic acid,and that gaps in similarity of up to 5% of the total number ofnucleotides in the reference sequence are allowed.

Whether the capture sequence of a capture probe and/or detector probewill hybridize to the target sequence of a target oligonucleotidedepends on the degree of complementarity between the target sequence andthe capture sequence, as well as both the hybridization conditions andthe stringency of the wash after hybridization. As used herein, thephrase “conditions that permit hybridization” refers to hybridizationparameters, as well as wash parameters, that permit hybridizationbetween two oligonucleotides, as are understood in the art. For example,conditions that permit hybridization include, but are not limited to,more stringent hybridization and wash conditions, such as incubation at42° C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing with 0.1×SSC at about 65° C., 68° C. or70° C. Of course, hybridization and wash conditions can be set to alower stringency. Lower stringency hybridization and wash conditionsinclude, but are not limited to, incubation at 42° C. in a solutioncomprising 30% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in a solution of 2×SSC or 1×SSC or 0.5×SSC at about55° C. or 60° C. or 65° C. As is within the capacity of one of ordinaryskill in the art, the conditions to permit hybridization can be easilyand routinely optimized to require a lower or higher degree ofcomplementarity between a capture probe and/or detector probe and atarget nucleic acid before hybridization will occur. For example,anionic detergents such as sodium dodecyl sulfate (SDS) may be used toenhance the stringency of hybridization or washing, and exclusionmolecules such as PEG may be used to increase the effectiveconcentration of reaction components.

A capture probe or detector probe may be an oligonucleotide or apolynucleotide, as these terms are understood in the art. A captureprobe or detector probe may be, for example, at least 10, 15, 20, 25,30, 35, 40, 45, 50, 75, 100, 150, 200, 300, 400, 500 or 750 nucleotidesin length. For convenience, the term “oligonucleotide” as used hereinencompasses all of these lengths. In some embodiments, capture probesand/or detector probes may be up to and including about 2000 nucleotidesin length. In some embodiments, capture probes and/or detector probesare about 15 to about 60 nucleotides in length.

The length of the capture probe and/or detector probe and the capturesequence(s) thereof and the conditions of hybridization may be tailoredto form a specific complex with the nucleic acid of the intended target.The stability of a hybrid complex, commonly measured by its meltingtemperature, is related to the concentration of the probe, thehybridization conditions, the length of the hybrid complex and thedegree of sequence identity between the capture sequence and the targetsequence. The stability of a hybrid complex is decreased by mismatchesand is increased by the number of base pairs in the hybrid complex. Suchrelationships are detailed, for example, in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, 3^(rd) ed., Cold Spring Harbor LaboratoryPress, New York (2001), which is herein incorporated by reference. Aprobe can be used to distinguish between closely related sequences thatdiffer in the hybrid complex by as little as a single base. That is, aprobe may be perfectly matched with a target nucleic acid from onespecies of microorganism but may differ in sequence by one base with thenucleic acid target of a second species, for example. Under theappropriate conditions, the presence of the mismatch causes the probe toform a hybrid complex with one target but not with the other. Becausethe relative difference in melting temperature between the matched andmismatched complexes decreases with increasing size of the probe, theprobe preferably should be 25 bases in length or less to detect a singlemismatch. See Sambrook et al. While longer probes can be used todiscriminate between closely related targets, the targets preferablyshould diverge to a progressively greater extent as the size of theprobe increases.

After a hybrid complex is formed, the methods of the present inventionthen detect the hybrid complex. If a hybrid complex is detected, thepresence of the hybrid complex indicates that the target sequence wasreleased from a microorganism in the sample tested. Accordingly, thepresence of a hybrid complex indicates the presence in the sample of themicroorganism and/or epidemiological marker containing the targetsequence. In contrast, when a hybrid complex is not detected, theabsence of the hybrid complex indicates that the target sequence was notreleased from a microorganism in the sample tested. Accordingly, theabsence of a hybrid complex indicates that the sample most likely doesnot contain the microorganism and/or epidemiological marker containingthe target sequence.

Any method of detecting the hybrid complex can be used in the presentinvention, provided that one of skill in the art can rely on thedetection methods to identify the presence of a hybrid complex. In someembodiments, at least one detector probe, in addition to the captureprobe, is used to detect the hybrid complex. The detector probehybridizes to a single-stranded portion (or “target sequence”) of thetarget nucleic acids that are captured.

A detector probe may comprise a reporter group to facilitate detection.As used herein, a “reporter group” means an entity that can generate adetectable signal. A reporter group may be incorporated into a detectorprobe, a reporter group may be directly linked or bound to a detectorprobe, or a reporter group may be indirectly linked or bound to adetector probe. As explained in more detail hereinabove, as an exampleof an indirect attachment using a linker molecule, a detector probe maycomprise a reporter adapter sequence linker, which is capable of bindingvia hybridization to a reporter probe, which is bound to a reportergroup. Detector probes that “comprise” reporter groups include detectorprobes that have a reporter group, reporter adapter sequence linkers,and the like incorporated therein, as well as detector probes that aredirectly or indirectly linked or bound to reporter groups, reporteradapter sequence linkers, and the like.

Many different reporter groups are known in the art. For example,radioactive isotopes may be used as reporter groups. Radioactiveisotopes may be, for example, incorporated into or attached to adetector probe, thereby generating a radioactive probe. Examples of aradioactive isotopes that can be used as reporter groups include, butare not limited to ³²P, ³³P¹³¹I, ⁹⁰Y, ¹⁸⁸Re, ¹⁸⁶Re, ⁶⁷Cu, ¹⁹⁸Au, ¹⁰³Pdand ²¹²Pb/²¹²Bi.

Enzymes and/or enzyme systems that can be used to generate a detectablesignal may also be used as reporter groups. Enzymes and/or othercomponents of enzyme systems can be attached directly to a detectorprobe or can attach indirectly to a detector probe. For example, adetector probe may be biotinylated. As a specific, non-limiting example,a detector probe may be bound to BioTEG, which is biotin with a 15 atomtetra-ethyleneglycol spacer. Biotin possesses the ability to bind toavidin or streptavidin. Continuing the example, an enzyme such ashorseradish peroxidase would be conjugated to avidin or streptavidin,thereby allowing the horseradish peroxidase to localize to the hybridcomplex via binding of the biotin of the biotinylated detector probe andthe avidin of the avidin-enzyme conjugate. Upon addition of a substrate,horseradish peroxidase would then generate a detectable colorimetricsignal as is readily understood in the art. Additional examples ofenzymes that may be used as reporter groups include, but are not limitedto alkaline phosphatase, glucose oxidase, β-galactosidase, soybeanperoxidase and luciferase.

Fluorescent or other detectable molecules may also be used as reportergroups and may be attached directly or indirectly to the detector probe.Non-limiting examples of detectable molecules that may be used asreporter groups include, but are not limited to, fluorescein,fluorescein isothiocyanate (FITC), rhodamine red, ROX™ (Invitrogen,California, USA), Cy™ dyes (Amersham, New Jersey, USA), Bodipy™ dyes(Molecular Probes, Oregon, USA), TAMRA™ dyes (Molecular Probes, Oregon,USA), TET™ (Molecular Probes, Oregon, USA), Texas Red® (MolecularProbes, Oregon, USA), europium dyes, chromogenic moieties and greenfluorescent protein (GFP).

Reporter groups may also comprise an “amplification sequence,” i.e., anucleotide sequence that can initiate nucleic acid amplification. Forexample, an amplification sequence may be appended to a detector probe,or an amplification sequence may be an integral part of a detectorprobe. An amplification sequence can initiate nucleic acid replication,or amplification, using any form of amplification including, but notlimited to, strand displacement amplification (SDA), polymerase chainreaction (PCR), reverse transcriptase-strand displacement amplification(RT-SDA), reverse transcriptase-polymerase chain reaction (RT-PCR),nucleic acid sequence based amplification (NASBA), MessageAmp™amplification (Ambion, Inc., Texas, USA), transcription-mediatedamplification (TMA), rolling circle amplification, and Qβ replicaseamplification. The nucleic acids may be amplified prior to contactingthem with a capture probe or after they are contacted with the captureprobe.

Instead of or in addition to the use of a detector probe, a hybridcomplex may also be detected using such standard methods asnon-specifically labeling the hybrid complex. For example, intercalatingdyes, such as ethidium bromide, may be used to label the hybrid complexfor detection. Other examples of non-specific labeling of the hybridcomplex include, but are not limited to, acridine orange, SYBR™ GreenI/II (Molecular Probes, Oregon, USA), SYBR Gold, propidium iodide andcyanine monomers or dimers.

In some embodiments, the methods and/or probes of the present inventionare used to monitor the efficacy of antimicrobial patient therapy bysequential sampling of specimens over time. The detection of mRNA hasbeen correlated with microbial viability. Hellyer et al., J. Clin.Microbiol. 37: 290-295 (1999). Accordingly, the detection of RNA in afirst sample, followed by administration of antimicrobial therapy andsubsequent failure to detect RNA in a second sample, most likelyindicates a successful response to therapy. In contrast, detection ofRNA in the second sample would most likely indicate the continuedpresence of viable organisms in the specimen, and the need for continuedtherapy or a change in therapeutic regimen.

In some other embodiments, the methods and/or probes of the presentinvention are used to quantify the number of organisms in a samplethrough the use of an internal standard and by comparison of signalintensities with controls. The internal standard may be RNA or DNA thatis free in solution or encapsulated, such as in an Armored RNA™ (AmbionDiagnostics, Texas, USA) particle or recombinant bacterium, to protectagainst degradation. The internal standard may be seeded into the sampleat any point prior to detection. In some embodiments, the internalstandard is seeded in the sample prior to concentration and lysis of themicroorganism(s). The internal standard is detected using specificcapture probes that permit distinction of the standard from targetnucleic acid and from other nucleic acids that may be present. Thesignal generated by the internal standard in the test sample is comparedto that from controls that comprise different levels of the standardnucleic acid conjugated directly to the solid phase. By plotting a curveof signal intensities for the controls, the proportion of the internalstandard recovered from the specimen may be calculated. In similarfashion, controls comprising different levels of the target sequenceconjugated directly to the solid phase may be used to quantify theamount of target present in the processed sample. By correcting forrecovery of nucleic acid using the internal standard, the quantity oftarget nucleic acid in the original sample may then be calculated.

It is important to note that the methods of the present invention alsohave application outside the fields of human and animal infectiousdisease and are particularly suited to applications requiring rapid,sensitive and specific detection and/or identification of multipleanalytes. Accordingly, the methods of the present invention can be usedto detect microorganisms in samples in many fields including, asnon-limiting examples, therapeutic monitoring, food and environmentaltesting and monitoring deployment of weapons of bioterrorism.

The present invention also relates to kits for detecting the presence orabsence of at least one microorganism in a sample. The kits comprise asolid support, as defined hereinabove, comprising at least one captureprobe, also defined hereinabove. The at least one capture probe may be amicroorganism-specific probe, a probe that is for an epidemiologicalmarker, and/or a probe that is for a drug resistance marker. The kits ofthe present invention also comprise at least one reporter group, aspreviously described hereinabove. The kits may also comprise a vessel.The vessel can be used to collect or concentrate the sample and/or toisolate the nucleic acids released from the microorganisms. The vesselsmay also be used when amplifying the nucleic acids released from themicroorganism, if desired or necessary. Examples of vessels include, butare not limited to, evacuated blood collection tubes, eppendorf tubes,test tubes, etc. The kits may further comprise enzymes or otherchemicals, such as media, detergents, buffers, acids, bases, andchaotropes used to lyse the microorganisms present in the sample. Thereporter group(s) of the kits further comprises at least one detectorprobe. In such embodiments, the reporter group may be incorporated intoor onto the detector probe, as previously described herein. As with thecapture probe, the detector probe may be a universal probe or aspecies-specific probe. Kits of the present invention may also comprisepositive, negative, and/or internal controls. In certain embodiments,kits of the present invention may comprise a sufficient number of probesand/or amounts of other components to permit the performance of only asingle assay or group of related assays using probes according to thepresent invention. In other embodiments, kits of the present inventionmay comprise a sufficient number of probes and/or amounts of othercomponents to permit the performance of multiple assays using probesaccording to the present invention.

The Figures provide non-limiting exemplary illustrations of embodimentsof the present invention and devices and non-limiting exemplarysequences useful in practicing the present invention. One exemplaryembodiment of the probes and methods of the present invention isillustrated in FIG. 1. In FIG. 1, bacteria in blood are concentratedinto a volume of 300 μL using a Wampole isolator tube. Next, thebacteria are lysed to release RNA or DNA, and this RNA- and/orDNA-containing solution in either a crude or purified form is applied toa microarray solid support that has capture probes immobilized thereto.The capture probes may be, for example, microorganism-specific probes,probes that are for epidemiological markers, and/or probes that are fordrug resistance markers. In this particular embodiment, after themicrobial RNA and/or DNA is allowed to hybridize with the capture probesto form hybrid complexes, the complexes are detected using a detectorprobe that bears a reporter group.

Two exemplary embodiments of the use of the probes and methods of thepresent invention are illustrated in FIGS. 2A and 2B. The twoembodiments of FIG. 2 differ in the manner in which capture probes areimmobilized on a solid support. The capture probes of the embodimentillustrated in FIG. 2A are immobilized directly on the solid support.The capture probes are species-specific, and species A-specific captureprobes are immobilized to one spot (A), while species B-specific captureprobes are immobilized to a different spot (B). In a further embodiment,two or more capture probes may be immobilized on a single spot to permitthe capture and detection of two or more target sequences derived fromdifferent organisms in a single location.

In contrast, the capture probes of the embodiment illustrated in FIG. 2Bare immobilized indirectly on the solid support through the use ofimmobilization probes. Each species-specific capture probe comprises animmobilization sequence that is hybridized to an immobilization probe.The immobilization probe is immobilized to a spot on the solid support.Each of the immobilization probes of the embodiment illustrated in FIG.2B is specific for a specific type of capture probe. Immobilizationprobes A hybridize specifically to capture probes A (which are specificfor species A), and immobilization probes B hybridize specifically tocapture probes B (which are not shown, but which are specific forspecies B). Capture probe A-specific immobilization probes areimmobilized to one spot (A), while capture probe B-specificimmobilization probes are immobilized to a different spot (B). In afurther embodiment, two or more immobilization probes may be immobilizedon a single spot to permit the capture and detection of two or moretarget sequences derived from different organisms in a single location.

In each of the embodiment of FIG. 2A and the embodiment of FIG. 2B, afirst target sequence of a target oligonucleotide released frommicroorganism species A hybridizes to a species A-specific capturesequence on a species A-capture probe, forming a hybrid complex. Thehybrid complex is detected by allowing a second target sequence on thetarget oligonucleotide to hybridize to a species A-specific capturesequence on a species A-specific detector probe. A reporter adaptersequence on the detector probe is allowed to hybridize with a universalreporter probe having a reporter group attached thereto.

Immobilization probe-capture probe combinations may be varied andcustomized, however. As just one non-limiting example, using FIG. 2B forillustrative purposes, the immobilization probes specific for speciesA-specific capture probes could also bind to capture probes specific fora second species (“species C”). Species C could be, for example, of thesame genus as species A. In such a case, species A-specific captureprobes and species C-specific capture probes would comprise the sameimmobilization sequences, while having capture sequences specific forspecies A or C, respectively.

FIG. 3 depicts an exemplary solid support that may be used to immobilizeprobes of the present invention and may be used in the methods and kitsof the present invention. The solid support illustrated in FIG. 3 is aflow-through microarray chip, which has the advantage of increasing therate of hybridization between the capture probe and the RNA and/or DNAof the microorganism. The flow-through chip illustrated in FIG. 3 wasproduced by MetriGenix, Inc. (Maryland, USA).

FIG. 4 depicts a vessel capable of concentrating the microorganisms in asample, such as blood, that can be used in the methods and kits of thepresent invention.

FIGS. 5A-C illustrate embodiments of the invention in whichimmobilization probes comprising sequences from the human genes K-alpha(tubulin alpha-1), PPIA (peptidylprolyl isomerase A), and UBC(ubiquitin-conjugating enzyme E2A) are immobilized onto differentregions (or “spots”) of a solid support. Each of the immobilizationprobes is hybridized to an oligonucleotide capture probe comprising (1)an immobilization sequence complementary to the immobilization probe and(2) and an organism-specific capture sequence. The capture sequences ofthe capture probes are designed to hybridize to sequences that arespecific for E. coli, S. aureus or S. epidermidis. An E. coli, S. aureusor S. epidermidis target oligonucleotide is bound to each capture probevia hybridization between a capture sequence of the capture probe and afirst target sequence of the target oligonucleotide.

A biotinylated detector probe is bound to each target oligonucleotidevia hybridization between a capture sequence of the detector probe and asecond target sequence of the target oligonucleotide. Streptavidinconjugated to horseradish peroxidase enzyme is bound to the biotinmolecules of the biotinylated detector probes. A horseradish peroxidaseenzyme substrate is added, and a detectable signal is generated.

FIG. 6 depicts synthetic target oligonucleotides derived fromdiscontiguous regions within the ssrA genes (small stable RNA A) of E.coli, S. aureus, and S. epidermidis. Target sequences in the targetoligonucleotides are underlined or boxed. Capture probes and detectorprobes that may be used to capture and detect these sequences are alsoshown, and their capture sequences are underlined or boxed.Specifically, regions of complementarity between capture probes andtarget nucleic acids are underlined, and regions of complementaritybetween detector probes and target nucleic acids are boxed. The captureprobes also comprise immobilization sequences from the human genesK-alpha (tubulin alpha-1), PPIA (peptidylprolyl isomerase A), and UBC(ubiquitin-conjugating enzyme E2A). These human gene sequences (SEQ IDNOs:59, 65, and 71) are italicized in FIG. 6 and set forth in Table 4a,hereinbelow. FIG. 6 is described in more detail in the exampleshereinbelow.

FIG. 7F depicts the arrangement of immobilization probes and controls onchips according to the invention. FIGS. 7A-E depict results fromexemplary assays using probes and methods according to the invention.FIGS. 7A-F are described in more detail in the examples hereinbelow.

FIG. 8A depicts the arrangement of capture probes and controls on chipsaccording to the invention. FIGS. 8B-E depict results from exemplaryassays using probes and methods according to the invention. FIGS. 8A-Eare described in more detail in the examples hereinbelow.

The following experimental examples are provided to illustrate certainembodiments of the invention, but are not intended to limit theinvention. The examples and embodiments described herein areillustrative, but not limiting, of the probes, methods and kits of thepresent invention. Other suitable modifications and adaptations of thevariety of conditions and parameters normally encountered in typicallaboratory and which are obvious to those skilled in the art are withinthe spirit and scope of the invention described herein.

Example 1 Determination of the Specific Gravities of E. Coli, S. Aureusand C. Albicans in Comparison to Whole Human Blood

To prepare a density gradient, 60% iodixanol (1.32 g/ml) was dilutedwith Dulbecco's phosphate buffered saline to densities of 1.080, 1.101,1.121 and 1.143 g/ml. A 2-ml volume of each of the density solutions wassequentially added to a conical centrifuge tube in order of density,starting with the highest.

A 1.25-ml volume of anticoagulated whole human blood was diluted 4-foldwith Dulbecco's phosphate-buffered saline. The diluted blood wasinoculated with E. coli, S. aureus or C. albicans at a finalconcentration of 2500 organisms/ml. The organism-spiked blood wasoverlayered on the density gradient. The gradient was centrifuged at3000×g for 20 minutes at ambient temperature. Following centrifugation,2 ml fractions were removed from the density gradient. A 0.1-ml volumeof each fraction was pipetted onto a blood agar plate and streaked fororganism isolation. Each plate was incubated for approximately 18 hoursat 35° C. under ambient air.

E. coli and S. aureus were isolated from the fractions with densities ofbetween 1.101 and 1.121 and 1.121 and 1.143 g/m, respectively. C.albicans was isolated from the fraction with a density of 1.143 g/ml.Blood cells were observed in the fraction with a density between 1.080and 1.101 g/ml. The latter data are consistent with reports in theliterature, in which the density of blood is estimated to be 1.090-1.101g/ml. These data demonstrate that organisms can be differentiallyseparated from blood in a density solution provided that their specificgravity is higher than that of blood.

Example 2 Differential Separation of S. Aureus and C. Albicans fromBlood in a Density Solution

A 5-ml volume of anticoagulated whole human blood was inoculated with S.aureus or C. albicans at a final concentration of 2500 organisms/ml.After inoculation, a 2-ml volume was overlayered on a 3-ml volume ofdensity solution at 1.090 g/ml, prepared by dilution of 60% (1.32 g/ml)iodixanol with Dulbecco's phosphate-buffered saline. The densitysolution was centrifuged at 5445×g for 40 minutes at ambienttemperature. After centrifugation, 1-ml fractions were collected fromthe density solution. The organisms were isolated from the densityfractions as described in Example 1. In contrast, the blood cells wereobserved to remain at the top of the density solution.

These data demonstrate that both S. aureus and C. albicans can beseparated from whole blood by centrifugation through a density matrix.These organisms can be further processed to purify protein, DNA or RNAand be used in downstream molecular or microbiological applications.

Example 3 Differential Separation of E. Coli, S. Aureus and C. Albicansfrom Lysed Human Blood

A 5-ml volume of whole human blood was lysed with Triton X-100 at afinal concentration of 1%. The detergent-treated blood was inoculatedwith E. coli, S. aureus or C. albicans at a final concentration 2500organisms/ml. Sixty percent iodixanol (1.32 g/ml) was diluted to 1.090g/ml with Dulbecco's phosphate-buffered saline. A 3-ml volume of densitysolution at 1.090 g/ml was overlayered with a 2-ml volume of lysed bloodcontaining the three organisms. The density solution was centrifuged andfractions were collected as described in Examples 1 and 2. The organismswere isolated from the fractions as described in Example 1. A11 threeorganisms were recovered from the bottom of the density solution.Meanwhile, the debris from the lysed blood cells was observed at the topof the density solution.

These data show that E. coli, S. aureus and C. albicans can be separatedefficiently from blood that has been lysed in Triton X-100. As describedin Example 2, recovered organisms can be used in subsequent downstreammolecular or microbiological applications.

Example 4 Detection of E. Coli-, S. Aureus-, and S. Epidermidis-SpecificOligonucleotides Using MGX® Universal Chips

The following example demonstrates the detection of E. coli-, S. aureus-and S. epidermidis-specific synthetic oligonucleotides using theMetriGenix® 4D™ DNA chip.

MetriGenix® chips (MetriGenix, Inc., Maryland, USA) were spotted withimmobilization probes of approximately 60 nucleotides in lengthcomprising sequences from the human genes K-alpha (tubulin alpha-1) (SEQID NO:84), PPIA (peptidylprolyl isomerase A) (SEQ ID NO:85), and UBC(ubiquitin-conjugating enzyme E2A) (SEQ ID NO:86). The sequences of theimmobilization probes (SEQ ID NOs:84-86) are set forth in Table 4b. Thelocations of the immobilization probes immobilized on the chips areshown in FIG. 7F. Also included on each chip were negative (“buffer”)and positive (“staining”) control spots that were prepared by spottingof buffer or immobilization of a biotinylated beta-actin-derivedoligonucleotide, respectively. The “buffer” spots served as negativecontrols for non-specific hybridization. The “staining” spots served aspositive staining controls for the chemiluminescent detection reaction.The sequence of the positive staining control biotinylatedbeta-actin-derived oligonucleotide (SEQ ID NO:83) was as follows: 5′-CCCAGG GAG ACC AAA AGC-Biotin. Each of the chips used in this example(Chips A-E) bore the indicated capture probes in the arrangementindicated in FIG. 7F.

Each of the immobilization probes was hybridized to an oligonucleotidecapture probe comprising (1) an immobilization sequence of approximately30 bases complementary to the immobilization probe and (2) and anorganism-specific ssrA gene capture sequence of about 20-nucleotides.The sequences of the capture probes (SEQ ID NOs:55, 61, 67) are setforth in Table 2 and in FIG. 6.

The capture sequences of the capture probes were designed to hybridizeto sequences that are specific for E. coli, S. aureus, or S.epidermidis. The capture sequences (SEQ ID NOs:1, 6, 10) of the captureprobes are underlined in FIG. 6 and set forth in Table 3. The captureprobe specific for E. coli also comprises an immobilization sequence(SEQ ID NO:59; italicized in FIG. 6 and set forth in Table 4a)complementary to the K-alpha immobilization probe. The capture probespecific for S. aureus also comprises an immobilization sequence (SEQ IDNO:65; italicized in FIG. 6 and set forth in Table 4a) complementary tothe PPIA immobilization probe. The capture probe specific for S.epidermidis also comprises an immobilization sequence (SEQ ID NO:71;italicized in FIG. 6 and set forth in Table 4a) complementary to the UBCimmobilization probe. (See FIGS. 5A-C for a schematic illustration.)

TABLE 2 Sequences of Capture Probes Used in Example 4 SEQ Target TargetID Organism Gene 5′-3′ Probe Sequence NO: E. coli ssrATCC TCG GTA CTA CAT GCT 55 TAG TAC ATT CAA CAG AATCCA CAC CAA CCT CCT CAT A S. aureus ssrA TTG ATT AAG TTT CTT CTA 61AAC AGA TAC ATC ATA ATC ATA AAC TTA ACT CTG CAA TCC A S. epidermidisssrA CAG TTA TAT TTA ACC GAA 67 ATG TGT ACA GAA AGT GCAATG AAA TTT GTT GAA ACC TTA

TABLE 3 Capture Sequences of Capture Probes Used in Example 4 SEQ TargetTarget ID Organism Gene 5′-3′ Probe Sequence NO: E. coli ssrATCC TCG GTA CTA CAT GCT 6 TAG S. aureus ssrA TTG ATT AAG TTT CTT CTA 1AAC AGA S. epidermidis ssrA CAG TTA TAT TTA ACC GAA 10 ATG TGT

TABLE 4a Immobilization Sequences of Capture Probes Used in Example 4Immobilization Capture Sequence Probes' Corresponds Target to Portion ofSEQ Organism Human Gene ID and Gene Sequence 5′-3′ Probe Sequence NO:E. coli K-alpha TTC AAC AGA ATC CAC 59 ssrA ACC AAC CTC CTC ATAS. aureus PPIA TCA TAA TCA TAA ACT 65 ssrA TAA CTC TGC AAT CCA S. UBCGAA AGT GCA ATG AAA 71 epidermidis TTT GTT GAA ACC TTA ssrA

TABLE 4b Immobilization Probes Used in Example 4 Used to ImmobilizeImmobilization Probe Capture Probe Corresponds to Portion SEQSpecific for Target of Human Gene ID Organism and Gene Sequence 5′-3′Probe Sequence NO: E. coli ssrA K-alpha CGT GAA GAT ATG GCT 84GCC CTT GAG AAG GAT TAT GAG GAG GTT GGT GTG GAT TCT GTT GAAS. aureus ssrA PPIA ATG TTT TCC TTG TTC 85 CCT CCC ATG CCT AGCTGG ATT GCA GAG TTA AGT TTA TGA TTA TGA S. epidermidis ssrA UBCTGG TCC TGC GCT TGA 86 GGG GGG GTG TCT AAG TTT CCC CTT TTA AGGTTT CAA CAA ATT TCA TTG CAC TTT C

To mimic the presence of microorganism-derived target oligonucleotidesin a sample, synthetic target oligonucleotides comprising first targetsequences complementary to the capture sequences of the capture probesspecific for each organism were used to flood the MetriGenix® chip. Thesequences of the synthetic target oligonucleotides (SEQ ID NOs:54, 60,66) are set forth in Table 5 and in FIG. 6. The synthetic targetoligonucleotides were bound to (or “captured by”) the organism-specificimmobilization probes via hybridization between the capture sequence ofthe capture probe and the first target sequence of the synthetic targetoligonucleotide. (See FIGS. 5A-C for a schematic illustration.) Thesequences of the first target sequences (SEQ ID NOs:57, 63, 69) of thesynthetic target oligonucleotides are underlined in FIG. 6 and set forthin Table 6.

TABLE 5 Sequences of Synthetic Target Oligonucleotides Used in Example 4SEQ ID Organism Gene 5′-3′ Target Oligonucleotide Sequence NO: E. colissrA TCC CTA GCC TCC GCT CTT AGG ATA 54 AAG ACT GAC TAA GCA TGT AGT ACCGAG GAT S. aureus ssrA AAG TCT GTT TAG AAG AAA CTT AAT 60CAA ACT AGC ATC ATG TTG GTT GTT TAT CAC TTT TCA TGA TGC S. epidermidisssrA AAC ACA TTT CGG TTA AAT ATA ACT 66 GAC AGT ATC ATG TTG GTT GTT TATTGC TTA GCA TGA TGC GA

TABLE 6 First Target Sequences of Synthetic TargetOligonucleotides Used in Example 4 SEQ ID Organism Gene 5′-3′Target Oligonucleotide Sequence NO: E. coli ssrACTA AGC ATG TAG TAC CGA GGA 57 S. aureus ssrATCT GTT TAG AAG AAA CTT AAT CAA 63 S. epidermidis ssrAACA CAT TTC GGT TAA ATA TAA CTG 69

The synthetic target oligonucleotides also comprised second targetsequences (SEQ ID NOs:58, 64, 70; boxed in FIG. 6 and set forth in Table7) that were complementary to the organism-specific capture sequences of5′-biotinylated detector probes. The sequences of the detector probes(SEQ ID NOs:56, 62, 68) are set forth in Table 8 and in FIG. 6. The 5′biotin labels attached to the detector probes are also shown in Table 8and in FIG. 6. Detection of a captured synthetic target oligonucleotideoccurred through hybridization between the second target sequence of thesynthetic target oligonucleotide (SEQ ID NOs:58, 64 or 70) and thecapture sequence of the detector probe specific for that targetoligonucleotide (SEQ ID NOs:1, 6 or 10). Therefore detector probesspecific for captured target oligonucleotides were immobilized. Unbounddetector probes were removed by washing. Streptavidin—linked horseradishperoxidase and an appropriate chemiluminescent substrate (Luminol) werethen added. The streptavidin—linked horseradish peroxidase was bound toimmobilized biotin-bearing detector probes via bonding betweenstreptavidin and biotin. In the presence of hydrogen peroxide,horseradish peroxidase catalyzes the oxidation of the substrate Luminol,resulting in the emission of light that is captured by a Charge-CoupledDevice (CCD) camera. (See FIGS. 5A-C for a schematic illustration.) Thecapture sequences (SEQ ID NOs:2, 7, 8) of the detector probes are boxedin FIG. 6 and set forth in Table 9.

TABLE 7 Second Target Sequences of Synthetic TargetOligonucleotides Used in Example 4 SEQ ID Organism Gene 5′-3′Target Oligonucleotide Sequence NO: E. coli ssrA TAG CCT CCG CTC TTA GGA58 S. aureus ssrA GGT TGT TTA TCA CTT TTC ATG A 64 S. epidermidis ssrATTG TTT ATT GCT TAG CAT GAT GC 70

TABLE 8 Sequences of Detector Probes Used in Example 4 SEQ ID NO:  Target Target labeled with Organism Gene 5′-3′ Probe Sequence biotinE. coli ssrA Biotin-CAC TAC GAC TCT CGG 56 TCT GAT TCT ATT TGC TCC TAAGAG CGG AGG CTA S. aureus ssrA Biotin-CAC TAC GAC TCT CGG 62TCT GAT TCT ATT TGC TCA TGA AAA GTG ATA AAC AAC C S. epidermidis ssrABiotin-CAC TAC GAC TCT CGG 68 TCT GAT TCT ATT TGC CAT CATGCT AAG CAA TAA ACA A

TABLE 9 Capture Sequences of Detector Probes Used in Example 4 SEQTarget Target ID Organism Gene 5′-3′ Probe Sequence NO: E. coli ssrATCC TAA GAG CGG AGG CTA 7 S. aureus ssrA TCA TGA AAA GTG ATA AAC 2 AAC CS. epidermidis ssrA CAT CAT GCT AAG CAA TAA 8 ACA A

The sample mix loaded onto to each of the chips used in this example(Chips A-E) contained 10 nM organism-specific capture probe, 100 nMbiotinylated organism-specific detector probe, and 50 nM targetoligonucleotide. The sample mix containing the target oligonucleotidesand probes was heated on a 95° C. heat block for five minutes andimmediately cooled on ice for two minutes. Reagents were flowedsequentially through MetriGenix® chips as follows using an MGX® 2000hybridization station (MetriGenix, Inc., Maryland, USA):

-   -   a. 2500 μL Buffer 1 (saline sodium phosphate-EDTA (SSPE)        containing Triton X-100) at a flow rate of 500 μL/min;    -   b. Blocking Reagent (buffered goat serum); 6 min at a flow rate        of 10 μL/min;    -   c. 1250 μL Buffer 2 (morpholinoethane sulfonic acid buffer (MES)        containing formamide, EDTA, sarcosine and NaCl) at a flow rate        of 500 μL/min;    -   d. Hybridization Mixture (Buffer 1 containing 10 nM capture        probe; 100 nM detector probe; 50 nM target oligonucleotide); 2        hours at a flow rate of 10 μL/min;    -   e. 2000 μL Buffer 1 at a flow rate of 500 μL/min;    -   f. Blocking Reagent; 6 min at a flow rate of 20 μL/min;    -   g. 1000 μL Buffer 1 at a flow rate of 500 μL/min;    -   h. Staining Reagent (streptavidin-conjugated horseradish        peroxidase in a solution containing NaH₂PO₄, EDTA, NP40 and        Tween-20)    -   i. 2000 μL Buffer 1 at a flow rate of 500 μL/min. Substrate        (Luminol).

All steps were performed at ambient temperature with the exception ofthe 2 hour hybridization incubation that was conducted at 37° C. Imagesof the arrays were captured on an MGX® 1200CL Detection Station using aCCD camera.

TABLE 10 Combinations of Targets and Detector Probes Tested FigureShowing Target Chip Results Oligonucleotides Capture Probes DetectorProbes A FIG. 7A SEQ ID NOs: 54, 60 SEQ ID NOs: 55, 61 SEQ ID NOs: 56,62 and 66 and 67 and 68 B FIG. 7B None SEQ ID NOs: 55, 61 SEQ ID NOs:56, 62 and 67 and 68 C FIG. 7C SEQ ID NO: 54 SEQ ID NOs: 55, 61 SEQ IDNOs: 56, 62 and 67 and 68 D FIG. 7D SEQ ID NO: 60 SEQ ID NOs: 55, 61 SEQID NOs: 56, 62 and 67 and 68 E FIG. 7E SEQ ID NO: 66 SEQ ID NOs: 55, 61SEQ ID NOs: 56, 62 and 67 and 68

Results are depicted in FIGS. 7A-E. On Chip A (FIG. 7A), E. coli-, S.aureus- and S. epidermidis-based DNA oligonucleotides were detectedsimultaneously. Chip B (FIG. 7A) comprised a negative control withouttarget oligonucleotides and yielded only low background signals. ChipsC, D and E (FIGS. 7C-E, respectively) demonstrated specific detection ofeach of the three pathogenic organisms independently. There was nocross-reaction between capture and detection systems for any of thethree organisms, thereby demonstrating the ability to discriminate thesespecies using the probes and methods of the invention.

Example 5 A Prophetic Example of Specific Detection of E. Coli, S.Aureus, and S. epidermidis from Whole Blood Using ssrA or Rnase P RNAsas Targets

Sample processing: A 10-ml volume of anticoagulated whole human blood isseeded with 10,000 organisms each of S. aureus, S. epidermidis and E.coli and is treated with 1% (v/v) Triton X-100 for 10 minutes at ambienttemperature. A density matrix is formed by diluting iodixanol (1.32g/ml) to a density of 1.090 g/ml with Dulbecco's phosphate-bufferedsaline. The entire volume of lysed blood containing the aforementionedorganisms is overlayered onto 15 ml of the density matrix. The organismsare separated from blood debris by centrifugation at 5440×g for 40minutes at ambient temperature. At the end of the centrifugation step,the density matrix is decanted, and the resulting organism pellet isre-suspended in 100 μL of RNase-free water prior to the isolation oftotal RNA. The recovery of three organisms at this stage may be verifiedby growth on differential media, followed by biochemical identification.

Target Preparation: Total RNA from 100 μL of the bacterial suspension isprepared using a QIAGEN® RNeasy® kit (QIAGEN, GmbH). Total bacterial RNAis recovered and re-suspended in a final volume of 60 μL of RNase-freewater. Optionally, the RNA is fragmented in a fragmentation buffer (40mM Tris, 100 mM potassium acetate, 30 mM magnesium acetate, pH 8.0) at95° C. for 30 minutes to reduce secondary structure of the RNA.

Analysis On MetriGenix® Chips: Custom DNA chips comprising specific DNAcapture probes for ssrA or RNaseP RNAs from each of the targetorganisms, as well as other species of potential interest, aremanufactured by MetriGenix (MGX®, MetriGenix, Inc., Maryland, USA). Thehybridization and detection process may take place on an MGX® 2000hybridization station. Each capture probe comprises a target-specificregion (capture sequence) of about 20 bases in length, which isimmobilized on the surface of the chip via a 5′ 9-base linker that hasthe sequence TTT TAA AAT (SEQ ID NO:87). Capture probes for each targetorganism are focused in discrete areas of the chip (“spots”) permittingspecific detection and identification of each species present within asample. Negative controls for non-specific hybridization are included inwhich only a phosphate-buffered saline solution is spotted on the chip.Biotin-labeled DNA oligonucleotides are also spotted directly on thesurface to act as positive staining controls for the chemiluminescentdetection reaction. As depicted in FIG. 5, biotin-labeled detectionprobes are about 50 nucleotides in length and are positioned downstream,i.e., 3′ of the capture probes. Detection probes are mixed with varyingamounts of the fragmented total RNA at a final concentration of 140 nMin hybridization buffer (4×SSPE, 2.5×Denhardt's solution, with orwithout 30% formamide, pH 7.7). The probe-target RNA mixture isincubated at 95° C. for five minutes and then placed in a 45° C.water-bath for 10 minutes before applying a total volume of 66 μL to thechip surface, which is pre-blocked with goat serum to reducenon-specific binding. The hybridization process occurs at 4° C., or roomtemperature, for 10 hours at a flow rate of 10 μL/minute.Streptavidin-horseradish peroxidase solution (1.25 μg/μL) is then usedto flood the chip. The streptavidin molecules bind to the biotin labelson the detection probes that are, in turn, bound to the captured RNAtarget sequences. Unbound materials are removed by washing with 4×SSPE,pH 7.7.

Detection: Specific capture of the target RNA is visualized bychemiluminescence using an MGX® 1200CL Detection Station. In thepresence of hydrogen peroxide, horseradish peroxidase catalyzes theoxidation of the substrate Luminol, resulting in the emission of lightthat is captured by a CCD camera. The signal is collected and analyzedby MetriSoft™ software that corrects the signal intensity against thelocal background.

Predicted Results: No signals are detected in spots corresponding tonegative controls, while staining controls yield strong positivesignals. Positive signals are also detected at positions correspondingto the specific organism(s) present in the original sample. There is nosignal above background from spots corresponding to organisms not seededinto the original blood sample, i.e., spots corresponding to organismsother than S. aureus, S. epidermidis or E. coli.

Example 6 Specific Detection of Bacterial RNA Using Probes for 16S rRNA,tmRNA (ssrA Transcripts), and RNase-P Transcripts

The following example demonstrates the specific detection of E. coli andS. aureus RNA using the MetriGenix MGX™ 4D™ DNA chip (MetriGenix, Inc.,Maryland, USA).

Preparation Of Chips: MetriGenix® chips were spotted with capture probesof 27-33 nucleotides in length that were directed towards (1) transcriptsequences of the E. coli, S. aureus, or S. epidermidis ssrA gene (whichencodes transfer-messenger RNA (“tmRNA”)); (2) transcript sequences ofthe E. coli, S. aureus, or S. epidermidis Ribonuclease P (“RNase P” or“rnp”) gene; (3) E. coli 16S rRNA, (4) Trichomonas vaginalis 18S rRNA,or (5) Candida albicans 18S rRNA. In addition to an organism-specificcapture sequence, each capture probe comprised a 9-mer immobilizationsequence of 5′-TTT TAA AAT (SEQ ID NO:87), through which the captureprobe was attached to the chip surface. The sequences of the captureprobes, including the 5′ immobilization sequences, are set forth inTable 11. The capture sequences of the capture probes are set forth inTable 12.

The locations of the capture probes immobilized on the chips are shownin FIG. 8A. As can be seen in FIG. 8A, in certain locations (labeled“buffer”), only buffer was spotted on the chip. These locations servedas negative controls for non-specific hybridization. In other locations,(labeled “staining”), biotin-labeled DNA oligonucleotides were alsospotted directly on the surface to act as positive staining controls forthe chemiluminescent detection reaction. The sequence of the stainingcontrol oligonucleotide (SEQ ID NO:83) was as follows: 5′-CCC AGG GAGACC AAA AGC-Biotin. Each of the chips used in this example (Chip Nos.1-4) bore the indicated capture probes in the arrangement indicated inFIG. 8A.

TABLE 11 Probe Target Target SEQ ID Name Organism Gene 5′-3′Probe Sequence NO: CP1 E. coli ssrA TTT TAA AAT TCC TCG GTA 72CTA CAT GCT TAG CP2 S. aureus ssrA TTT TAA AAT TTG ATT AAG 73TTT CTT CTA AAC AGA CP3 S. epidermidis ssrA TTT TAA AAT CAT CAT GCT 74AAG CAA TAA ACA A CP4 E. coli rnp TTT TAA AAT GCA CTG GTC 75 GTG GGT TTCCP5 S. aureus rnp TTT TAA AAT TTA CTC TAT 76 CCA TAT CGA AAG ACT CP6S. epidermidis rnp TTT TAA AAT CTA TTC TAA 77 CCA TAT CCA ATG ACT CP7E. coli 16S rRNA TTT TAA AAT AGT GTG GCT 78 GGT CAT CCT CP8 Trichomonas18S rRNA TTT TAA AAT ATC CTG AAA 79 vaginalis GAC CCG AAG CCT GTC CP9Candida albicans 18S rRNA TTT TAA AAT TTG TTC CTC 80GTT AAG GTA TTT ACA TTG TAC TC

TABLE 12 Capture Sequences of Capture Probes Used in Example 6 Part ofTarget Target SEQ ID Probe Organism Gene 5′-3′ Probe Sequence NO: CP1E. coli ssrA TCC TCG GTA CTA CAT GCT TAG 6 CP2 S. aureus ssrATTG ATT AAG TTT CTT CTA AAC 1 AGA CP3 S. epidermidis ssrACAT CAT GCT AAG CAA TAA ACA A 8 CP4 E. coli rnp GCA CTG GTC GTG GGT TTC46 CP5 S. aureus rnp TTA CTC TAT CCA TAT CGA AAG 47 ACT CP6S. epidermidis rnp CTA TTC TAA CCA TAT CCA ATG 48 ACT CP7 E. coli16S rRNA AGT GTG GCT GGT CAT CCT 52 CP8 Trichomonas 18S rRNAATC CTG AAA GAC CCG AAG CCT 81 vaginalis GTC CP9 Candida 18S rRNATTG TTC CTC GTT AAG GTA TTT 82 albicans ACA TTG TAC TC

Application Of Target RNA: Total RNA was isolated from E. coli, S.aureus, and S. epidermidis using a QIAGEN®-based extraction protocol.RNA (1.5 μg) from each organism was added either alone or in combinationto buffer containing 1×SSPE, 0.15M NaCl, 0.01M NaH₂PO₄, 0.001M EDTA),2.5% Triton X-100 and used to flood replicate MetriGenix® chipsessentially as described in Example 4. Hybridization took place over 10hours at room temperature at a flow rate of 10 μL/min. The combinationsin which the RNA was used are set forth below and in Table 13. Chipswere washed twice with MES buffer containing 0.88M NaCl, 0.02M EDTA,0.5% sarcosine, 33% formamide prior to staining.

Detection Of Captured RNA: Organism-specific 50-mer detector probes thatwere labeled at the 3′ end with BioTEG (Biotin with a 15 atomtetra-ethyleneglycol spacer). The detector probes were washed over thechips in combinations set forth below and in Table 13. The sequences ofthe detector probes are set forth in Table 14. The 3′ BioTEG labelsattached to the detector probes are also shown in Table 14.Streptavidin-horseradish peroxidase solution (1.25 pg/μL) was then usedto flood the chip. The streptavidin molecules bound to the biotin labelson the detection probes that were, in turn, bound to the captured RNAtarget sequences. Unbound materials were removed by washing with 1×MES.Bound detector probes were visualized by chemiluminescence.Specifically, a chemiluminescent substrate (Luminol) was used to floodthe chips. In the presence of hydrogen peroxide, horseradish peroxidasecatalyzed the oxidation of the substrate Luminol, resulting in theemission of light that was captured by a CCD camera. The assays wereperformed on an MGX® 2000 hybridization station (MetriGenix, Inc.,Maryland, USA). Images of the array were captured over a 10 secondperiod using an MGX® 1200CL Detection Station (MetriGenix, Inc.,Maryland, USA) equipped with a CCD camera. Results are depicted in FIGS.8B-E.

TABLE 13 Combinations of Targets and Detector Probes Tested in Example 6Figure Showing Target Detector Probes Contacted with RNA from Chip No.Results Organisms Target Organism Immobilized on Chip 1 FIG. 8B E. coliEco_ssrA_DP50 (specific for E. coli ssrA) Eco_rnp_DP50 (specific for E.coli rnp) Eco_16_DP50 (specific for E. coli 16S rRNA) 2 FIG. 8C S.aureus Eco_ssrA_DP50 (specific for E. coli ssrA) S. epidermidisEco_rnp_DP50 (specific for E. coli rnp) Eco_16_DP50 (specific for E.coli 16S rRNA) 3 FIG. 8D S. aureus Sau_ssrA_DP50 (specific for S. aureusssrA) Sau_rnp_DP50 (specific for S. aureus rnp) 4 FIG. 8E E. coliSau_ssrA_DP50 (specific for S. aureus ssrA) S. epidermidis Sau_rnp_DP50(specific for S. aureus rnp)

TABLE 14 Sequences of Detector Probes Used in Example 6 SEQ ID NO:  Target Target labeled with Probe Name Organism Gene 5′-3′ Probe SequenceBioTEG^(‡) Eco_ssrA_DP50 E. coli ssrA TCA GTC TTT ACA TTC GCT 43TGC CAG CTG CGG ACG GAC ACG CCA CTA ACA AA- BioTEG Sau_ssrA_DP50S. aureus ssrA CTT CAA ACG GCA GTG TTT 44 AGC ATA TCC TAT TAA GGTTGA ATC GCG TTA AC- BioTEG Eco_rnp_DP50 E. coli rnpCCC CCC AGG CGT TAC CTG 49 GCA CCC TGC CCT ATG GAG CCC GGA CTT TCC TC-BioTEG Sau_rnp_DP50 S. aureus rnp TAG GAT ATT TCA TTG CCG 50TCA AAT TAA TGC CTT GAT TTA TTG TTT CAT CA- BioTEG Eco_16_DP50 E. coli16S CTC AGA CCA GCT AGG GAT 53 rRNA CGT CGC CTT GGT GAG CCGTTA CCC CAC CAA CA- BioTEG ^(‡)BioTEG = 3′ Biotin with a 15 atomtetra-ethyleneglycol spacer

FIG. 8B is a CCD image depicting Chip No. 1 after performance of theassay described in this example. Chip No. 1 was flooded with total RNAfrom E. coli. After washing to remove unbound RNA, the immobilized RNAon the chip was contacted with detector probes specific for E. colissrA, rnp and 16S rRNA transcript sequences (Eco_ssrA_DP50 (SEQ IDNO:43), Eco_Rnp_DP50 (SEQ ID NO:49), and Eco_(—)16_DP50 (SEQ ID NO:53)).As can be seen in FIG. 8B, only the spots on the chip corresponding tothe E. coli-specific target sequences and the positive staining controlsyielded signals above background.

FIG. 8C is a CCD image depicting Chip No. 2 after performance of theassay described in this example. Chip No. 2 was flooded with total RNAfrom S. aureus and S. epidermidis. After washing to remove unbound RNA,the RNA immobilized on the chip was contacted with detector probesspecific for E. coli ssrA, rnp and 16S rRNA transcript sequences(Eco_ssrA_DP50 (SEQ ID NO:43), Eco_Rnp_DP50 (SEQ ID NO:49), andEco_(—)16_DP50 (SEQ ID NO:53)). As can be seen in FIG. 8C, only thespots on the chip corresponding to the positive staining controlsyielded signals above background.

FIG. 8D is a CCD image depicting Chip No. 3 after performance of theassay described in this example. Chip No. 3 was flooded with total RNAfrom S. aureus. After washing to remove unbound RNA, the immobilized RNAon the chip was contacted with detector probes specific for S. aureusssrA and rnp transcript sequences (Sau_ssrA_DP50 (SEQ ID NO:44) andSau_Rnp_DP50 (SEQ ID NO:50)). As can be seen in FIG. 8D, only the spotson the chip corresponding to the S. aureus-specific target sequences andthe positive staining controls yielded signals above background.

FIG. 8E is a CCD image depicting Chip No. 4 after performance of theassay described in this example. Chip No. 4 was flooded with total RNAfrom E. coli and S. epidermidis. After washing to remove unbound RNA,the immobilized RNA on the chip was contacted with detector probesspecific for S. aureus ssrA and rnp transcript sequences (Sau_ssrA_DP50(SEQ ID NO:44) and Sau_Rnp_DP50 (SEQ ID NO:50)). As can be seen in FIG.8E, only the spots on the chip corresponding to the positive stainingcontrols yielded signals above background.

For all combinations studied, no signals were detected in spotscorresponding to negative controls, while staining controls yieldedstrong positive signals. Strong positive signals were also detected atspots bearing immobilized RNA derived from a given specific organismwhen detector probes specific for that organism were contacted to theimmobilized RNA on the chip. No signals were detected at spots bearingRNA derived from a given specific organism when no detector probesspecific for that organism were contacted to the immobilized RNA on thechip. Contacting RNA derived from a given specific organism withdetector probe(s) specific for different organism(s) produced nosignals. These data demonstrate that both the capture probes and thedetector probes used were able to selectively bind to specific targetRNA. There was no cross-reaction between capture and detection systemsfor any of the three organisms, thereby demonstrating the ability todiscriminate these species using the probes and methods of theinvention.

There is no signal above background from spots corresponding toorganisms not seeded into the original sample, i.e., spots correspondingto organisms other than S. aureus, S. epidermidis or E. coli.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents.

1. A method for identifying the presence of at least one microorganismin a sample, the method comprising: (a) releasing RNA or DNA from the atleast one microorganism in the sample; (b) contacting the RNA or DNAwith at least one capture probe capable of hybridizing to a first targetsequence of the RNA or DNA, wherein the contacting is performed underconditions that permit hybridization between the first target sequenceand the at least one capture probe to form a microorganism-capture probehybrid complex, and wherein the at least one capture probe comprises atleast one sequence selected from the group consisting of SEQ IDNOs:1-53, 55, 56, 61, 62, 67, 68, and 72-78; and (c) detecting thepresence of the microorganism-capture probe hybrid complex by (i)contacting the RNA or DNA with at least one detector probe capable ofhybridizing to a second target sequence of the RNA or DNA, wherein thedetector probe comprises at least one reporter group and wherein thecontacting is performed under conditions that permit hybridizationbetween the second target sequence and the at least one detector probeto form a microorganism-capture probe-detector probe hybrid complex, andwherein the at least one detector probe also comprises at least onesequence selected from the group consisting of SEQ ID NOs:1-53, 55, 56,61, 62, 67, 68, and 72-78; and (ii) detecting the microorganism-captureprobe-detector probe hybrid complex by detecting the at least onereporter group wherein the presence of the microorganism-captureprobe-detector probe hybrid complex indicates the presence of the atleast one microorganism.
 2. The method of claim 1, wherein the firsttarget sequence and the second target sequence comprise the samesequence.
 3. The method of claim 1, wherein the capture probe isimmobilized on a solid support before hybridizing to the first targetsequence.
 4. The method of claim 1, wherein the microorganism-captureprobe hybrid complex is immobilized on a solid support.
 5. The method ofclaim 1, wherein the microorganism-capture probe-detector probe hybridcomplex is immobilized on a solid support.
 6. The method of claim 1,wherein the solid support is selected from the group consisting of latexbeads, agarose beads, paramagnetic beads, ferric oxide, microarraychips, filter paper, nitrocellulose filters, nylon membranes, glassslides and cellular membranes.
 7. The method of claim 6, wherein thesolid support is a microarray chip.
 8. The method of claim 1, whereintwo or more capture probes are immobilized on a single spot of the solidsupport.
 9. The method of claim 1, further comprising an immobilizationprobe that is capable of hybridizing to the capture probe to beimmobilized onto the solid support.
 10. The method of claim 1, whereinthe reporter group is selected from the group consisting of aradioactive isotope, an enzyme, a fluorescent molecule and anamplification sequence.
 11. The method of claim 10, wherein theamplification sequence initiates an amplification reaction selected fromthe group consisting of strand displacement amplification (SDA),polymerase chain reaction (PCR), reverse transcriptase-stranddisplacement amplification (RT-SDA), reverse transcriptase-polymerasechain reaction (RT-PCR), nucleic acid sequence based amplification(NASBA), transcription-mediated amplification (TMA), rolling circleamplification and Qβ replicase amplification.
 12. The method of claim 1,wherein detection of the microorganism-capture probe-detector probehybrid complex is accomplished via non-specifically labeling the hybridcomplex.
 13. A method for identifying the species of one or moremicroorganisms in a sample, the method comprising: (a) releasing RNA orDNA from the at least one microorganism in the sample; (b) contactingthe RNA or DNA with at least one species-specific capture probe capableof hybridizing to a first target sequence of the RNA or DNA, wherein thecontacting is performed under conditions that permit hybridizationbetween the first target sequence and the at least one species-specificcapture probe to form a species-specific microorganism-capture probehybrid complex, and wherein the at least one species-specific captureprobe comprises at least one sequence selected from the group consistingof SEQ ID NOs:1-53, 55, 56, 61, 62, 67, 68, and 72-78; and (c) detectingthe presence of the species-specific microorganism-capture probe hybridcomplex by (i) contacting the RNA or DNA with at least one detectorprobe capable of hybridizing to a second target sequence of the RNA orDNA, wherein the detector probe comprises at least one reporter groupand wherein the contacting is performed under conditions that permithybridization between the second target sequence and the at least onedetector probe to form a species-specific microorganism-captureprobe-detector probe hybrid complex, and wherein the at least onedetector probe also comprises at least one sequence selected from thegroup consisting of SEQ ID NOs:1-53, 55, 56, 61, 62, 67, 68, and 72-78;and (ii) detecting the species-specific microorganism-captureprobe-detector probe hybrid complex by detecting the at least onereporter group wherein the presence of the species-specificmicroorganism-capture probe-detector probe hybrid complex indicates thepresence of the at least one microorganism belonging to the species. 14.The method of claim 13, wherein the first target sequence and the secondtarget sequence comprise the same sequence.
 15. The method of claim 13,wherein the species-specific capture probe is immobilized on a solidsupport before hybridizing to the first target sequence.
 16. The methodof claim 13, wherein the species-specific microorganism-capture probehybrid complex is immobilized on a solid support.
 17. The method ofclaim 13, wherein the species-specific microorganism-captureprobe-detector probe hybrid complex is immobilized on a solid support.18. The method of claim 13, wherein the solid support is selected fromthe group consisting of latex beads, agarose beads, paramagnetic beads,ferric oxide, microarray chips, filter paper, nitrocellulose filters,nylon membranes, glass slides and cellular membranes.
 19. The method ofclaim 18, wherein the solid support is a microarray chip.
 20. The methodof claim 13, wherein two or more species-specific capture probes areimmobilized on a single spot of the solid support.
 21. The method ofclaim 13, further comprising an immobilization probe that is capable ofhybridizing to the species-specific capture probe to be immobilized ontothe solid support.
 22. The method of claim 21, wherein the reportergroup is selected from the group consisting of a radioactive isotope, anenzyme, a fluorescent molecule and an amplification sequence.
 23. Themethod of claim 22, wherein the amplification sequence initiates anamplification reaction selected from the group consisting of stranddisplacement amplification (SDA), polymerase chain reaction (PCR),reverse transcriptase-strand displacement amplification (RT-SDA),reverse transcriptase-polymerase chain reaction (RT-PCR), nucleic acidsequence based amplification (NASBA), transcription-mediatedamplification (TMA), rolling circle amplification, and Qβ replicaseamplification.
 24. The method of claim 13, wherein detection of themicroorganism-capture probe-detector probe hybrid complex isaccomplished via non-specifically labeling the hybrid complex.
 25. Amethod of determining the efficacy of an antimicrobial patient therapy,comprising: (a) identifying the presence or absence of a microorganismin a first patient sample according to the method claim 1; and (b)identifying the presence or absence of the microorganism in a secondpatient sample according to the method of claim 1; wherein the firstpatient sample and the second patient sample are taken sequentially overtime, and wherein detection of the microbial nucleic acid in the firstsample and subsequent failure to detect nucleic acid in the secondsample indicates a successful response to therapy; and detection of themicrobial nucleic acid in the second sample indicates the continuedpresence of viable organisms in the sample.
 26. The method of claim 25,wherein the solid support is selected from the group consisting of latexbeads, agarose beads, paramagnetic beads, ferric oxide, microarraychips, filter paper, nitrocellulose filters, nylon membranes, glassslides and cellular membranes.
 27. The method of claim 25, wherein thesolid support is a microarray chip.
 28. The method of claim 25, whereintwo or more capture probes are immobilized on a single spot of the solidsupport.
 29. The method of claim 25, further comprising animmobilization probe that is capable of hybridizing to the capture probeto be immobilized onto the solid support.
 30. The method of claim 25,wherein the reporter group is selected from the group consisting of aradioactive isotope, an enzyme, a fluorescent molecule and anamplification sequence.
 31. The method of claim 30, wherein theamplification sequence initiates an amplification reaction selected fromthe group consisting of strand displacement amplification (SDA),polymerase chain reaction (PCR), reverse transcriptase-stranddisplacement amplification (RT-SDA), reverse transcriptase-polymerasechain reaction (RT-PCR), nucleic acid sequence based amplification(NASBA), transcription-mediated amplification (TMA), rolling circleamplification, and Qβ replicase amplification.
 32. The method of claim25, wherein detection of the microorganism-capture probe-detector probehybrid complex is accomplished via non-specifically labeling the hybridcomplex.
 33. A kit for detecting the presence or absence of at least onemicroorganism in a sample, comprising: (a) a solid support; (b) at leastone capture probe comprising at least one capture sequence capable ofhybridizing to at least one target sequence of RNA and/or DNA from themicroorganism to form a microorganism-capture probe hybrid complex;wherein the at least the detector probe also comprises at least onesequence selected from the group consisting of SEQ ID NOs:1-53, 55, 56,61, 62, 67, 68, and 72-78; (c) at least one detector probe capable ofhybridizing to a second sequence of the RNA or DNA, wherein the detectorprobe comprises at least one reporter group, and wherein the detectorprobe comprises at least one sequence selected from the group consistingof SEQ ID NOs:1-53, 55, 56, 61, 62, 67, 68, and 72-78; and (d) a vesselto collect, concentrate, amplify or isolate the RNA or DNA.
 34. The kitof claim 33, wherein the vessel is selected from the group consisting ofevacuated blood collection tubes, eppendorf tubes and test tubes. 35.The kit of claim 34, wherein the solid support is selected from thegroup consisting of latex beads, agarose beads, paramagnetic beads,ferric oxide, microarray chips, filter paper, nitrocellulose filters,nylon membranes, glass slides and cellular membranes.
 36. Anoligonucleotide for use in detecting a microorganism selected from thegroup consisting of Staphylococcus aureus, Escherichia coli,Staphylococcus epidermidis, Klebsiella pneumoniae, Enterococcusfaecalis, Pseudomonas aeruginosa, Streptococcus pneumoniae,Streptococcus mutans, Streptococcus gordonii, Clostridium perfringens,Clostridium botulinum, Haemophilus influenzae, Enterococcus durans,Streptococcus pyogenes, Streptococcus agalacticae, Clostridium difficileand Enterococcus faecium.
 37. The oligonucleotide of claim 36, whereinStaphylococcus aureus is selected from the group consisting of SEQ IDNOs:1, 2, 44, 47, 50, 61, 62, 73 and
 76. 38. The oligonucleotide ofclaim 36, wherein Escherichia coli is selected from the group consistingof SEQ ID NOs:3-7, 43, 46, 49, 52, 53, 55, 56, 72, 75 and
 78. 39. Theoligonucleotide of claim 36, wherein Staphylococcus epidermidis isselected from the group consisting of SEQ ID NOs:8-10, 45, 48, 51, 67,68, 74 and
 77. 40. The oligonucleotide of claim 36, wherein Klebsiellapneumoniae is selected from the group consisting of SEQ ID NOs:11-13.41. The oligonucleotide of claim 36, wherein Enterococcus faecalis isselected from the group consisting of SEQ ID NOs:14-16.
 42. Theoligonucleotide of claim 36, wherein Pseudomonas aeruginosa, is selectedfrom the group consisting of SEQ ID NOs:17 and
 18. 43. Theoligonucleotide of claim 36, wherein Streptococcus pneumoniae isselected from the group consisting of SEQ ID NOs:19 and
 20. 44. Theoligonucleotide of claim 36, wherein Streptococcus mutans is selectedfrom the group consisting of SEQ ID NOs:21 and
 22. 45. Theoligonucleotide of claim 36, wherein Streptococcus gordonii is selectedfrom the group consisting of SEQ ID NOs:23 and
 24. 46. Theoligonucleotide of claim 36, wherein Clostridium perfringens is selectedfrom the group consisting of SEQ ID NOs:27 and
 28. 47. Theoligonucleotide of claim 36, wherein Clostridium botulinum is selectedfrom the group consisting of SEQ ID NOs:29 and
 30. 48. Theoligonucleotide of claim 36, wherein Haemophilus influenzae is selectedfrom the group consisting of SEQ ID NOs:31 and
 32. 49. Theoligonucleotide of claim 36, wherein Enterococcus durans is selectedfrom the group consisting of SEQ ID NOs:35-37.
 50. The oligonucleotideof claim 36, wherein Streptococcus pyogenes is selected from the groupconsisting of SEQ ID NOs:38-40.
 51. The oligonucleotide of claim 36,wherein Streptococcus agalacticae is selected from the group consistingof SEQ ID NOs:41 and
 42. 52. The oligonucleotide of claim 36, whereinClostridium difficile is selected from the group consisting of SEQ IDNOs:25 and
 26. 53. The oligonucleotide of claim 36, wherein Enterococcusfaecium is selected from the group consisting of SEQ ID NOs:33 and 34.